1 Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore
117609
2 MRC Centre for Developmental Neurobiology, King's College London, New Hunts
House, Guy's Campus, London SE1 1UL, UK
* Authors for correspondence (e-mail: william.chia{at}kcl.ac.uk; mcbyangn{at}imcb.nus.edu.sg)
Accepted 28 November 2002
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Summary |
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Key words: Mouse Pins, pins, Asymmetric cell division, Neuroblasts, Drosophila
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Introduction |
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Previous studies have shown that partner of insc (pins)
(Parmentier et al., 2000;
Schaefer et al., 2000
;
Yu et al., 2000
), together
with insc (Kraut and
Campos-Ortega, 1996
; Kraut et
al., 1996
), bazooka (baz)
(Kuchinke et al., 1998
;
Schober et al., 1999
;
Wodarz et al., 1999
),
DmPar6 (Petronczki and Knoblich,
2001
), DaPKC (Wodarz
et al., 2000
) and G
i
(Schaefer et al., 2001
) form
an apically localized protein complex that plays a crucial role in mediating
NB asymmetric divisions. These studies have provided insight into how this
apical protein complex might be assembled. During delamination NBs inherit
apical-basal polarity cues through Baz/DaPKC/DmPar6, an evolutionarily
conserved protein cassette that is already apically localized in epithelial
cells of the neuroectoderm from which NBs arise. Insc, which can directly
interact with Baz in vitro, is then recruited to this complex during
delamination. Pins, which can directly interact with Insc, and G
i,
which can interact with Pins, then joins this complex. pins encodes a
protein with seven tetratricopeptide repeats (TPR) at its N-terminal portion
responsible for interaction with the asymmetric localization domain of Insc
(Yu et al., 2000
), and three
GoLoco motifs (also known as GPR motif) at its C-terminal region capable of
binding to the G
subunit of heterotrimeric G proteins
(Schaefer et al., 2000
;
Parmentier et al., 2000
). Pins
is required for the maintenance of Insc asymmetric localization and for Baz
stability. Maternal and zygotic depletion of pins results in the
cytoplasmic distribution of Insc during mitosis, similar to that observed in
NBs derived from baz germ line clones (GLCs). In addition loss of
pins function results in the drastic reduction in the intensity of
Baz apical crescents.
The participation of all the apical components is required for correct
mitotic spindle orientation along the apical basal axis, as well as the
asymmetric localization and segregation of the cell fate determinants Numb
(Uemura et al., 1989) and
Prospero (Pros) (Doe et al.,
1991
; Vaessin et al.,
1991
; Matsuzaki et al.,
1992
). Numb and Pros form cortical crescents at the basal side of
mitotic NBs and segregate to the basal GMC daughter following cytokinesis
(Hirata et al., 1995
;
Knoblich et al., 1995
;
Spana and Doe, 1995
). Pros
translocates into the nucleus of GMC where it activates GMC-specific gene
expression and represses NB-specific gene expression
(Doe et al., 1991
;
Vaessin et al., 1991
). Numb
can also be basally localized in mitotic GMCs and segregates preferentially to
one of the post-mitotic GMC daughter cells
(Buescher et al., 1998
). Numb
acts to antagonize Notch signaling by interacting with the intracellular
domain of Notch (Frise et al.,
1996
; Skeath and Doe,
1998
).
Several studies have shown that asymmetric cell divisions might be an
evolutionarily conserved phenomenon in Drosophila and vertebrates.
During ferret cortical neurogenesis, cortical precursors can undergo either
symmetric or asymmetric cell divisions
(McConnell, 1995). There is a
strong correlation between the nature of a precursor division and the
orientation of its cleavage plane. Symmetric divisions generating two
precursor daughters tend to occur when cleavage planes are oriented
perpendicular to the ventricular surface. By contrast, when cleavage planes
are aligned parallel to the ventricular surface, cortical precursors tend to
divide asymmetrically to generate a precursor and a neuron that migrates away
to the cortical plate (Chenn and McConnell,
1995
; Mione et al.,
1997
). Well-conserved homologues of Numb have been identified in
mouse (Zhong et al., 1996
),
rat (Verdi et al., 1996
) and
chicken (Wakamatsu et al.,
1999
). When ectopically expressed in Drosophila NBs and
sensory organ precursors (SOPs), numb is asymmetrically localized and can
functionally substitute for numb function. Moreover, vertebrate numb
homologues are asymmetrically localized in the ventricular zone progenitors of
the developing mouse CNS and in neuroepithelial cells of the chick. Similar to
its fly counterpart, asymmetrically localized numb physically interacts with
the cytoplasmic region of mammalian notch and antagonizes notch signaling.
Overexpression studies in mice and chicks suggest a role in neural
differentiation and proliferation for these vertebrate numb homologues
(Verdi et al., 1996
;
Wakamatsu et al., 1999
). A
putative mammalian homologue of pros, Prox1, has also been reported
(Oliver et al., 1993
;
Wigle and Oliver, 1999
).
We previously showed that Drosophila Pins shares high sequence
homology with human LGN (Yu et al.,
2000). It has also been reported that activator of G-protein
signaling 3 (AGS3) (Takesono et al.,
1999
; Peterson et al.,
2000
), a rat homologue of pins, acts as a guanine
dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP
by G
i3 (Bernard et al.,
2001
; De Vries et al.,
2000
; Natochin et al.,
2000
). However, how any of the mammalian Pins-related proteins
might relate functionally to the fly Pins remains unclear. Here, we report the
identification and characterization of a mouse homologue of pins.
Mouse Pins encodes a protein that shares high homology with Pins.
Pins mRNA is present from embryonic stage E11 onwards, in a variety
of tissues except skeletal muscle. In the CNS, Pins transcript is
associated with zones where proliferative cells are found. Like Pins
(Yu et al., 2002
), PINS can
also physically interact with the asymmetric localization domain of Insc
through its TPR (TPR3-TPR7). When expressed in Drosophila NBs, PINS
localizes as an apical cortical crescent and can substitute for Pins
functionally in all aspects of NB asymmetric division, such as Insc apical
localization, Numb/Pros basal localization and apical/basal spindle
orientation, as well as resolution of distinct sibling neuronal cell fates.
Deletion analysis of PINS shows that, like Pins, its C-terminal GoLoco
containing region specifies targeting to the membrane, whereas its N-terminal
TPR further refines localization to the apical cortex. Our results show that
Pins can be recognized by the Drosophila asymmetric division
machinery and functionally replace pins for all aspects of NB
asymmetric divisions.
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Materials and Methods |
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RNA and protein analyses
The cDNA from IMAGE:949074 corresponding to nucleotides 840-3355 of mouse
Pins were labeled and used to probe northern blots containing 2 µg
of poly(A)+ RNA isolated from different stages of mouse embryos and
a variety of mouse tissues (Clontech). For PINS detection, blots were
incubated with pre-immune serum (1:2500) and purified anti-PINS antibody
(1:2500), respectively, at 4° C overnight. Immunoreactive bands were
visualized using horseradish peroxidase-coupled donkey anti-rabbit
immunoglobulin G (IgG) secondary antibody and the ECL kit (Amersham).
GST fusion protein production and antiserum production
A polypeptide containing amino acids 478-672 of the mouse PINS coding
region was expressed as a GST fusion protein in Escherichia coli and
purified using glutathione sepharose (Pharmacia). Eluted GST-PINS protein was
used to immunize rabbits. Purification of anti-PINS antibody was performed as
follows: 200 µg of GST fusion protein was blotted on a membrane stripe.
Following overnight incubation with serum, the stripe was washed with PBS and
eluted by 1 M glycine (pH 2.5). Eluted antibody was neutralized using 1 M Tris
(pH 8.5).
Immunofluorescence and in situ hybridization
E12.5 and E15.5 mouse embryos were dissected in ice-cold PBS, and fixed
overnight in 4% paraformaldehyde in PBS. E15.5 brains were dissected before
cryoprotection, performed overnight at 4°C in PBS/30% sucrose. Brains and
embryos were quick frozen in OCT (Tissue-Tek) before sectioning (12 µm).
For in situ hybridization on sections, a 1 kb PstI fragment from
Pins (nucleotides 1460-2460) subcloned into pBluescriptSK
(Stratagene) was used as a template to generate sense and antisense RNA
probes. Sections were rinsed once in PBS and pre-hybridized for 30 minutes at
65°C in a hybridization solution bath. They were then incubated overnight
in an hybridization bath containing the probe. Hybridization procedure and
buffer composition used were essentially as described by Strahle et al.
(Strahle et al., 1994). For
immunofluorescence, tissue sections were rinsed once in PBS and blocked for 15
minutes in 1% BSA in PBT (0.25% Triton X-100 in PBS) and incubated with a
rabbit polyclonal anti-Ki-67 antibody (1:500 dilution in PBT-3% BSA) overnight
at 4°C. After three washes in PBT-0.1% BSA, an FITC-coupled goat
anti-rabbit IgG secondary antibody (Jackson ImmunoResearch) was applied for 2
hours in PBT-3% BSA. To visualize DNA, Hoechst 33258 (Sigma) was added in the
second of four washing baths (0.2 µg/ml in PBT). Sections were mounted in
Vectashield mounting medium (Vector Labs).
Drosophila embryo collections, heat-shock induction and
immunohistochemical staining were performed essentially as described
(Tio et al., 1999;
Yu et al., 2000
). For most
analyses, embryos were fixed in 4% paraformaldehyde for 15 minutes; for
tubulin staining, fixations were carried out in 37% formaldehyde for 3
minutes. After staining, embryos were mounted in vectashield (Vector Labs).
Primary antibodies used were rabbit anti-PINS (1:1000), anti-FLAG (M2, 1:2000,
Sigma), rabbit anti-Pins (1:1000), rabbit anti-Insc (1:1000), anti-Miranda
(1:1000, from F. Matsuzaki, RIKEN Center for Developmental Biology, Kobe,
Japan), rabbit anti-NUMB (1:1000) and anti-Pon (1:1000) (from Y. N. Jan,
Howard Hughes Medical Institute, UCSF, CA), mouse (1:15, from K. Zinn,
Caltech, Pasadena, CA) and rabbit anti-EVE (1:2000 from M. Frasch, Mount Sinai
School of Medicine, NY), mouse anti-Pros (1:5, from C. Q. Doe), rat
anti-ß-tubulin (1:15, Chemicon), mouse (1:2000, Promega) and rabbit
anti-ß-Gal (1:5000, Cappel). Secondary antibodies obtained from Jackson
Laboratories were Cy3 conjugated goat anti-rabbit IgG, FITC-conjugated goat
anti-mouse and FITC-conjugated goat anti-rat. DNA was visualized by TO-Pro 3
(1: 5000, Molecular Probes).
Images were acquired and recorded using a Bio-Rad confocal microscope 1024 and processed using Adobe Photoshop.
Transposon construction, germline transformation and overexpression
studies
The cDNA fragments encoding full-length mouse PINS, N-PINS (aa 1-369) and
C-PINS (aa 366-672) were fused in-frame with a double-FLAG epitope at their
respective C-terminal ends. The resultant DNA fragments were cloned into the
hs-Casper transformation vector. Embryo injections used to generate
germline transformants were performed essentially as described
(Spradling, 1986). The
expression of PINS, N-PINS and C-PINS in embryos was induced by a 15 minute
heat shock at 34°C; after 1 hour recovery at 25°C in a moist chamber,
embryos were processed for immunohistochemistry. Induced PINS, N-PINS and
C-PINS possess two tandem FLAG epitopes at their C-terminus and were detected
using either rabbit anti-PINS (1:1000 dilution) or monoclonal antibody M2
against FLAG (1:2000, Sigma). For rescue experiments, hs-Pins,
pins89/TM3, Sb, Ubx-LacZ male flies were crossed to
pins89/pins89 females and embryos were
collected and subjected to heat shock. Mutant embryos were identified with
anti ß-gal staining and analyzed for PINS, Insc, Miranda, and Pon
localization. In parallel, the same collection of embryos were double stained
with rabbit anti-ß-Gal and anti-FLAG to ascertain that the observed
non-ß-Gal stained embryos were expressing the heat-induced transgene
products. The heat shock regime used for the rescue of the RP2 duplication
phenotype was previously described (Tio et
al., 1999
).
Yeast two-hybrid assay and protein-binding assays
Yeast two-hybrid assay and protein-binding assays were performed as
described (Yu et al.,
2000).
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Results |
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The overall amino acid sequence of PINS is 49% identical to that of
Drosophila Pins, 92% to that of human LGN, 60% to rat AGS3 and 45% to
worm PINS (Fig. 1B). Mouse PINS
is molecularly more similar to human LGN than rat AGS3, and another family of
ESTs has since appeared in mouse databases, showing higher homology to rat
AGS3 than to LGN (mouse AGS3). Hence, mouse PINS/LGN and m-AGS3/AGS3 may be
paralogues formed by duplication after divergence of mammals and flies. The
TPR of fly Pins have been shown to be required for binding to Insc, suggesting
that mouse PINS might also be able to interact with fly Insc or an
unidentified mouse homologue of Insc. PINS and LGN have four GoLoco motifs and
they also share high homology at their C-terminal portion with Pins,
suggesting that they may also be able to bind to the -subunit of
heterotrimeric G proteins. The overall homology among worm PINS, fly Pins,
mouse Pins, rat AGS3 and human LGN may reflect functional conservation during
evolution.
Mouse Pins is expressed widely in a variety of tissues and
its expression in the developing CNS is enriched in regions showing high
levels of proliferation
To examine the expression pattern of mouse Pins, we carried out a
series of northern blots. The developmental northern blot analysis showed a
major 3.3 kb transcript, detected from E11 to E17 embryos. No transcript was
detected in E7 embryos (Fig.
2B). Multiple-tissue northern blot analysis revealed that mouse
Pins mRNA was expressed in most adult tissues, including heart,
liver, kidney, brain, lung, spleen and testis, but not in skeletal muscle
(Fig. 2A).
pins plays an important role in the process of asymmetric division
of neural progenitors in both the central and peripheral nervous systems of
the fly. Given the sequence conservation between pins and
Pins, we speculated that Pins might have a function in
controlling some aspects of nervous system development in vertebrates as well,
and studied its expression pattern during neurogenesis. In situ hybridization
shows that Pins is expressed in the CNS of E12.5 mouse embryos. The
expression is restricted, however, to a population of cells lying next to the
ventricular zone (Fig. 3A-C).
Three days later, at E15.5, the transcript is detected in the ventricular zone
of the telencephalon and the roof of the diencephalon
(Fig. 3F), with very little
expression detected more caudally in the hindbrain and virtually no expression
in the spinal cord (not shown). The ventricular distribution suggests that
Pins could be restricted to mitotic progenitors. This would be
consistent with the limited expression observed in the anterior brain at
E15.5, when most neural progenitors are located in the developing cortex. We
assessed whether Pins expression is limited to mitotic cells or is
maintained for a certain period after cells exit the cell cycle and start to
differentiate. Pins and Ki-67, a nuclear antigen specific for
proliferating cells (Gerdes et al.,
1983), were detected in adjacent sections of E12.5 embryos. The
results show a restriction of pins transcript to cells in the
proliferative region of the CNS (Fig.
3C-C'', 3G-H''). These results suggest that in the CNS,
Pins is expressed in mitotic cells and absent from differentiating
cells. Mouse Pins transcript was also detected in ganglia of the PNS
(dorsal root ganglia, cranial sensory neurons and the sympathetic chain) at
E12.5 (Fig. 3D,E).
|
To determine the subcellular distribution of mouse Pins gene
product, we raised a rabbit polyclonal antibody against the C-terminal half
(aa 478-666) of PINS. In a western blot analysis, this purified antibody
specifically recognized a protein of 75 kDa in liver and brain extracts,
consistent with the predicted molecular weight of PINS, whereas the pre-immune
serum did not (Fig. 2C). In
parallel, polyclonal antibodies were raised against two specific peptides (aa
459-479 and aa 644-657), and an anti-LGN antibody directed against a peptide
conserved between LGN and PINS was obtained
(Blumer et al., 2002). Using
this panel of antibodies, we could not achieve consistent results on mouse
cryosections and were not able to conclusively address the question of PINS
subcellular distribution in vivo.
Mouse PINS can interact with Insc in the yeast two-hybrid and GST
pull-down assays
Because Pins interacts directly with Insc through its seven TPR
(Yu et al., 2002), we tried to
ascertain whether PINS also binds to Insc using yeast two-hybrid assays and in
vitro binding assays. We confirmed that PINS interacts with Insc through its
seven TPR; moreover, the minimal region including TPR3-7 is sufficient for its
interaction with Insc (Fig.
4A).
|
The PINS-Insc protein interaction was further confirmed by GST pull-down assays. Mouse PINS is able to bind to all GST-Insc fusion proteins (full-length Insc, aa 1-859; Insc-5, aa 288-497; Insc-2, aa 258-578) containing the asymmetric localization domain of Insc (aa 288-497) but not to GST alone nor to N-terminal Insc (N-Insc, aa1-330), which lacks the asymmetric localization domain (Fig. 4B). Furthermore, N-PINS (aa 1-369) and TPR3-7 (aa 129-369) were pulled down by GST-Insc but not GST alone, whereas C-PINS (aa 366-672) could not bind to GST-Insc (Fig. 4C,D). These results indicate that the region TPR3-7 of PINS can interact directly with Insc and that PINS mimicks the Insc-binding properties of fly Pins.
Mouse PINS is asymmetrically localized as an apical crescent when
ectopically expressed in Drosophila NBs
Because PINS can interact with Insc, we assessed whether the cellular
machinery responsible for the localization of Pins in Drosophila NBs
can recognize and localize PINS. Transgenic fly lines carrying P-element
insertions containing full-length PINS, N-PINS (aa 1-369) and C-PINS (aa
366-672) under the control of the hsp70 promoter were generated.
Those various PINS constructs were tagged at their extreme carboxyl termini
with a double-FLAG epitope and were detected in the transgenic animals with
anti-FLAG antibody following induction. No expression was detected in the
transgenic embryos lacking heat-shock induction
(Fig. 5C), whereas PINS is
ubiquitously expressed in embryos treated with 15 minute heat shock
(Fig. 5F). The subcellular
localization of ectopically expressed full-length PINS resembles that of the
endogenous fly Pins in NBs, forming an apical cortical crescent during mitosis
(Fig. 5F). By contrast, neither
N-PINS nor C-PINS were asymmetrically localized in NBs when overexpressed in a
wild-type (WT) background. C-PINS was cytoplasmic, whereas C-PINS was cortical
in mitotic (metaphase) NBs, showing identical localization patterns to their
equivalent counterparts from fly Pins (Fig.
5I,L) (Yu et al.,
2002). These results show that the protein localization machinery
of Drosophila NBs can recognize full-length PINS as if it were the
endogenous Drosophila Pins.
|
Surprisingly, although PINS and fly Pins show only 32% identity at their C-terminus, the anti-PINS antibody, which was raised against the C-terminal region, can recognize the endogenous fly Pins in wild-type Drosophila embryos (Fig. 5A). In Drosophila pins-mutant embryos, anti-PINS staining did not show any immunoreactivity (Fig. 5B), indicating that this cross-reaction is specific to fly Pins.
Mouse Pins can fulfill all aspects of Drosophila
pins function in neuroblast asymmetric divisions
Because PINS can interact physically with Insc and can localize as an
apical cortical crescent when ectopically expressed in the Drosophila
NBs, we wondered whether PINS could functionally substitute for
Drosophila Pins. To determine whether Pins can function in
flies, the phenotypes associated with flies lacking both maternal and zygotic
pins function were scored following hsp70-mediated
expression of full-length PINS, N-PINS and C-PINS, respectively.
In pins null mutants, Insc asymmetric localization is disrupted and the protein becomes cytoplasmic in dividing NBs. As expected, in transgenic pins mutant embryos without heat shock, Insc localization is cytoplasmic in dividing NBs (Fig. 5D). However, in pins mutant embryos with one copy of hs-Pins (full length) subjected to 15 minute heat shock and 1 hour recovery, the ectopically expressed full-length PINS was detected as an apical crescent using anti-FLAG antibody (Fig. 5F) and recruited Insc back to the apical cortex (Fig. 5G). By contrast, neither N-PINS (cytoplasmic; Fig. 5I) nor C-PINS (uniformly cortical; Fig. 5L) could form an apical crescent or restore Insc apical crescent in transgenic pins mutants (Fig. 5J,M). These results provide the first indication that full-length PINS can be recognized by the protein localization machinery of Drosophila NBs and that it can functionally mimic fly Pins.
It has been shown that, although the mitotic spindle of WT mitotic domain 9
cells undergo a 90° rotation to orient perpendicular to the surface of the
embryo, this rotation fails to occur in the absence of pins function
and, consequently, the spindles are aligned parallel to the embryo surface
(Yu et al., 2000)
(Fig. 6A). Ectopically
expressed full-length PINS in pins mutant embryos is able to restore
this 90° spindle rotation in the cells of mitotic domain 9
(Fig. 6B) due to its ability to
recruit Insc to the apical cortex and stabilize the apical complex. By
contrast, expressing either N-PINS or C-PINS in pins mutants does not
restore this 90° spindle reorientation (data not shown).
|
In pins mutants, proteins that normally localize as basal cortical
crescents in mitotic NBs at metaphase, such as Mir/Pros and Pon/Numb, are
mislocalized either as randomly placed cortical crescents or are localized
throughout the cell cortex (Yu et al.,
2000). However, the introduction of PINS into pins
mutants, which localizes as an apical crescent
(Fig. 6C), allows the basal
proteins such as Miranda to localize normally
(Fig. 6D). Finally,
pins mutant embryos show defects in the resolution of alternative
sibling cell fates. These defects are most easily seen by following the
GMC4-2a>RP2/RP2sib sublineage. In pins mutant embryos, RP2sib
adopts the fate of its sibling, the RP2 neuron, resulting in the duplication
of the Even-skipped (Eve) positive RP2 neurons in 60% of the hemisegments
(Fig. 6G)
(Yu et al., 2000
). Moreover, a
small proportion of the GMC4-2a cells are also mis-specified, resulting in the
loss of Eve-expressing RP2 neurons in 15% of the hemisegments. However,
ectopic expression of PINS in pins mutant embryos results in a
significant rescue of these cell-fate transformations
(Fig. 6H) (only 6% of
hemisegments show RP2 duplication, n=180), whereas neither N-PINS nor
C-PINS could mediate this rescue (data not shown).
Taken together, these results show that Pins can functionally substitute for fly pins and apparently fulfill all aspects of its function in neuroblast asymmetric divisions.
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Discussion |
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Two mouse homologues of Pins exist
Database searches of the mouse genome with the fly Pins amino acid sequence
identified EST clones that encode two Pins-like proteins with varying
homologies to Pins. The mouse protein showing a higher percentage of homology
to Drosophila Pins is referred to as PINS. PINS shows a higher level
of homology to human LGN than to rat AGS3
(Fig. 1). The second mouse
protein is more closely related to AGS3 than to LGN and is therefore referred
to as mouse AGS3 (IMAGE:5720527 and IMAGE:5685096). Hence, there are at least
two homologues of Drosophila Pins in mouse, PINS and mouse AGS3.
Similarly, the human genome project also identifies two Pins-like sequences,
LGN (previously identified by Mochizuki et al.
(Mochizuki et al., 1996)) and
AGS3 (previously identified in the rat by Takesono et al.
(Takesono et al., 1999
)).
Hence, PINS/LGN and mouse AGS3/AGS3 appear to be paralogues, formed by
duplication after divergence of mammals and flies.
The two Pins-like proteins identified in the mammalian genomes have
different features. In situ hybridization of mouse Pins and
Ags3 showed a distinct distribution in the neural tube: Pins
is enriched in a layer of cortical precursors, whereas Ags3 is
uniformly distributed in the neural tube, suggesting distinct roles for these
proteins during neurogenesis (X. Morin, unpublished). This is reminiscent of
the localization profiles of mouse numb and numb-like in the
neural tube of the mouse embryo (Zhong et
al., 1997).
Asymmetry machinery of Drosophila NBs can recognize mouse
PINS
We have shown that mouse PINS interacts specifically with the asymmetric
localization domain of Drosophila Insc via its TPR region. This is
identical to what we observed with fly Pins
(Yu et al., 2002), and
suggests that they are structurally similar. Ectopically expressed PINS
colocalizes with endogenous fly Pins and forms an apical crescent in NBs.
Moreover, ectopically expressed N-terminal PINS and C-terminal PINS show
cortical and cytoplasmic localization, respectively, in NBs, equivalent to the
data we obtained in our previous domain dissection analyses of fly Pins
(Yu et al., 2002
). These
observations suggest that PINS achieves its asymmetric localization by using
the same mechanism as fly Pins (see below).
Several lines of evidence support the view that PINS, when overexpressed in Drosophila NBs, can functionally substitute for fly Pins. First, like the C-terminal rgion of fly Pins, C-terminal PINS is localized at the cell cortex of WT NBs, and its overexpression in Pins- embryos can lead to the generation of two equal-sized NB daughter cells (F. Yu, unpublished). Second, Insc can interact with both PINS and fly Pins through their seven TPR in vitro, suggesting that, like fly Pins, PINS is able to form a complex with Insc to orient asymmetric cell divisions of NBs. Third, the N-terminal regions of both PINS and fly Pins are insufficient to localize to the apical cortex; the C-terminal regions by themselves are uniformly localized to the cortex, but they do not localize apically. These observations indicate that both fly Pins and PINS mediate their apical localization in two steps: membrane targeting mediated by the C-terminal region and apical recruitment involving the N-terminal portion. Finally, the introduction of PINS can rescue all aspects of asymmetric division defects seen in pins mutant NBs.
What is the role of mouse pins in vertebrates?
We showed in a previous study that Drosophila Pins plays a crucial
role in asymmetric cell divisions of NBs
(Yu et al., 2000). It is
asymmetrically localized as a crescent in NBs, GMCs, muscle progenitor cells
(F. Yu, unpublished) and sensory organ precursors (Bellaiche, 2001).
Drosophila Pins is also expressed in most other embryonic and larval
tissues, where it is distributed around the lateral cell cortex.
Interestingly, in the developing CNS, mouse Pins shows a restricted
expression pattern. It is restricted to zones of proliferation and is absent
from differentiating post-mitotic cells. However, to date, we have not been
able to observe asymmetric localization of PINS by immunostaining of either
tissue culture cells or the developing ventricular zone of E12.5 mouse embryos
(data not shown). This apparent difference of their localization modes between
fly Pins and mouse PINS may reflect non-conservation of the asymmetry
machinery between Drosophila and mouse. In this regard, many
components known to be asymmetrically localized in Drosophila
for example Insc, partner of numb (pon), and
miranda do not have apparent orthologues in mammals. The
homologue of pros, prox 1, albeit present in mammals, plays a
distinct role. Although the mouse counterpart of Drosophila Numb
shows asymmetric localization in cortical precursors, the mode of its
asymmetric localization is probably different, as the mammalian counterpart of
Drosophila pon, which directs asymmetric localization of Numb in
Drosophila, does not appear to exist. Finally, insc seems to
be unique to flies and absent from mammals and nematodes.
Pins and Pins-related proteins share in common their ability to bind
G proteins and exert a GDI function, thereby inhibiting the exchange of
GDP-bound for GTP-bound forms of G
. Although present in different
tissues, Pins and Pins-related proteins may mediate their functions by
regulating G-protein activity. It has been reported recently that human LGN
localizes to the cytoplasm at interphase and subsequently to the mitotic
spindle during mitosis, and interacts with NuMA and regulates mitotic spindle
organization (Du et al., 2001
).
Another study reports that human LGN localizes to the nucleus at interphase
and the midbody during cytokinesis in cultured cells
(Blumer et al., 2002
).
It has been reported that, in addition to asymmetric localization in
dividing precursors, mouse NUMB is also localized to the Golgi region, in
particular endosomes, clathrin-coated pits and vesicles. NUMB can interact
with the endocytic machinery, -adaptin and Eps15. A dominant negative
form of NUMB inhibits clathrin-mediated endocytosis, suggesting a role for
NUMB in endocytosis (Santolini et al.,
2000
). Whether PINS functions in this process remains to be
determined. Heterotrimeric G proteins are known to be involved in protein
trafficking, particularly endocytosis (reviewed by
Ferguson, 2001
), and a role
for PINS in this process seems to be reasonable. PINS might act as a GDI to
regulate G signaling, which, in turn, regulates protein trafficking. Further
studies are required to understand the various functions of Pins
during mouse development.
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Acknowledgments |
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References |
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