1 Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston,
MA 02115, USA
2 Department of Molecular and Cellular Physiology, Stanford University,
Stanford, CA 94305, USA
* Author for correspondence (e-mail: thomas.schwarz{at}tch.harvard.edu)
Accepted 20 October 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Drosophila, Polarity, Trafficking
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The exocyst complex, a set of eight proteins first identified from
secretory mutants in yeast (Novick et al.,
1980), are attractive candidates for mediating directed traffic.
Yeast cells use an anisotropic secretory apparatus for polarized growth at a
selected bud site (Field and Schekman,
1980
). While the bud is growing, there is almost no increase in
the surface area of the mother cell
(Waddle et al., 1996
),
indicating that all membrane addition occurs at the bud tip. Later, secretion
is redirected to the neck between mother and bud
(Tkacz and Lampen, 1972
). The
exocyst complex marks these areas of membrane addition, localizing to the bud
tip of a growing daughter cell and the bud neck at the time of cytokinesis
(Finger et al., 1998
;
Guo et al., 1999
;
Novick et al., 1995
).
Mutations in each member of the exocyst complex block the polarized
trafficking that allows the bud to grow, but do not disrupt bud site
selection. Thus, the exocyst complex in yeast may provide a model for the
directed membrane traffic of developing cells in higher organisms.
The components of the exocyst complex, Sec3, Sec5, Sec6, Sec8, Sec10,
Sec15, Exo70 and Exo84, are conserved from yeast to mammals
(Hsu et al., 1996;
Novick et al., 1995
). In
multicellular organisms, though less extensively studied, these proteins are
implicated in establishing cell polarity. In epithelial cells, E-cadherin
mediated adhesion is sufficient to initiate the segregation of apical from
basolateral membrane proteins (Vega-Salas
et al., 1988
; Wang et al.,
1990
). The exocyst localizes to these sites of adhesion and is
required for the polarized transport of proteins to the basolateral domain
(Grindstaff et al., 1998
).
Neurons generate their polarity by directing membrane traffic to growing
neurites and growth cones, and by sorting proteins differentially between the
axon and dendrites (Burack et al.,
2000
; Craig et al.,
1995
). The exocyst localizes to the tips of growing neurites and
is required for neurite extension (Hazuka
et al., 1999
; Murthy et al.,
2003
). The distribution of the exocyst in some systems thus
suggests that, as in yeast, it will play a necessary role in directing
membrane traffic to subcellular domains.
Oogenesis in Drosophila requires the establishment and maintenance
of cellular asymmetry within the developing oocyte, and provides a system in
which to study directed membrane traffic. Within the egg chamber, which
consists of 16 germline cells interconnected by ring canals and surrounded by
somatic follicle cells, membrane ligands, adhesion proteins and transmembrane
receptors are called upon to signal within particular domains of the cell
surface. These signals allow the oocyte to migrate to the posterior end of the
egg chamber, induce reorganization of the microtubule cytoskeleton, establish
thereby asymmetries within the oocyte, and induce differentiations of the
adjacent follicle cells (Queenan et al.,
1999; van Eeden and St
Johnston, 1999
). These events all rely on the directed trafficking
of proteins, including E-cadherin, Gurken and the EGFR, to the plasma
membrane, so as to establish polarity within the oocyte and its surrounding
cells. In addition, the localization of some non-membrane determinants of
polarity, such as Oskar, may be indirectly dependent on membrane trafficking
(Bretscher, 1996
;
Dollar et al., 2002
;
Jankovics et al., 2001
;
Ruden et al., 2000
).
We have used mutations in sec5, a core component of the exocyst complex, to investigate the role of the exocyst in membrane trafficking and in the generation of cell polarity within the Drosophila ovary. We find Sec5 is dynamically localized during oogenesis in a manner that corresponds with the changing needs of the egg chamber for directed membrane traffic. Furthermore, Sec5 is required both for growth of the germline cells and for membrane trafficking necessary for establishment of the anteroposterior axis and dorsoventral pattern.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Germline clones of sec5E10 and sec5E13 were generated by crossing y,w;FRT40 sec5/Cyo;UAS-FLP female flies to y,w;FRT40 ovoD/Cyo;nanos-Gal4/TM3, Sb males. Ovaries were dissected from females of genotype FRT40 sec5/FRT40 ovoD;UAS-FLP/nanos-Gal4. sec5E13 ovaries expressing UAS-nod-GFP, were dissected from females of genotype y,w hs-FLP/+;FRT40 sec5E13/FRT40 ovoD;UAS-nod-GFP/nanos-Gal4 and heat shocked 1 hour at 37°C each day during larval and pupal development. Follicle cell clones of sec5E10 or sec5E13 were generated by crossing y,w;FRT40 sec5/Cyo;UAS-FLP females to y,w, hs-FLP;FRT40 Ubi-GFP;T155-Gal4, UAS-FLP/+ males and heat shocking 1 hours at 37°C during larval development.
Immunocytochemistry and microscopy
Ovaries from 1- to 4-day-old females were dissected in PBS, and kept on
ice. Ovaries were fixed in 6:1 Heptane:FIX [FIX=4 vol H2O, 1 vol
Buffer B (100 mM potassium phosphate pH 6.8, 450 mM KCl, 150 mM NaCl, and 20
mM MgCl2), and 1 vol 37% Formaldehyde] for 15 minutes. All antibody
staining was carried out in PBS, containing 0.5% BSA, 0.1% Triton-X-100 and 5%
normal goat or donkey serum. The following stains and primary antibodies were
used: Texas Red-X phalloidin and Hoechst 33342 (Molecular Probes);
fluorescein-lycopersicon esculentum lectin (Vector Laboratories);
mouse anti-Gurken 1D12; mouse anti-Syntaxin 8C3; mouse anti-Orb 4H8 and mouse
anti-FasIII 7G10 (Hybridoma bank); rabbit anti-ßgal (ICN); mouse
anti-Sec5 22A2 (Murthy et al.,
2003); rat anti-DE-cadherin
(Oda et al., 1994
); rat
anti-Yolkless (Schonbaum et al.,
2000
); rabbit anti-Oskar
(Ephrussi et al., 1991
);
rabbit anti-Par-1 (Shulman et al.,
2000
); mouse anti-Dhc P1H4
(McGrail and Hays, 1997
); and
mouse anti-Bicaudal-D (Suter and Steward,
1991
). Secondary antibodies used were: FITC-goat anti-mouse; FITC
or TR-donkey anti-mouse; FITC or TR-donkey anti-rat; and FITC-goat anti-rabbit
(Jackson Laboratories).
To disrupt microtubules, colcemid (50 µg/ml) in yeast paste was fed to
adult females for 8-10 hours (Theurkauf
and Hazelrigg, 1998).
Confocal data were acquired as single images or image stacks of multi-tracked, separate channels with a Zeiss LSM 510 microscope. Three-dimensional projections of image stacks were made with the 3D Zeiss software package. Nomarski images were captured on a Nikon Eclipse E800.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Wild-type development of the egg chamber has been subdivided into a series
of 14 stages (King, 1970).
During stages 2-6, after the egg chamber exits the germarium, the 15 nurse
cells and one oocyte grow at similar rates. The oocyte occupies the
posterior-most position among the group of sixteen cells
(Fig. 1A). By stage 7, the
microtubule cytoskeleton within the oocyte reorients and the nucleus moves to
an anterior corner position, i.e. along the circular rim where the lateral and
anterior surfaces of the oocyte meet, and thereby specifies this region as the
dorsal side (Fig. 1B).
Thereafter, the oocyte grows disproportionately to the nurse cells
(Fig. 1C).
|
Additionally, sec5E10 mutant germlines displayed
polarity defects. Normally, the oocyte occupies the posterior-most position,
owing to E-cadherin-based differential adhesion between the oocyte and the
posterior follicle cells (Godt and Tepass,
1998; Gonzalez-Reyes and St
Johnston, 1998
). In sec5E10 germlines, the
oocyte, labeled with antibodies to Dynein Heavy Chain (Dhc), was often
mispositioned (Fig.
1K,K'). In addition, the polar cells, which are important
for establishing initial polarity cues within the egg chamber
(Grammont and Irvine, 2002
),
are often mis-positioned within the heterozygous follicle epithelium
(Fig. 1L,M). In
sec5E10 germline clones, we also observed the development
of compound follicles with multiple germline cysts enclosed within a single
follicle epithelium (data not shown), a phenotype common to mutants that cause
a loss of polar cell identity. These phenotypes indicate that sec5 is
important for the initial establishment of anteroposterior polarity within the
egg chamber.
Females with germlines homozygous for sec5E13 lay eggs with dorsal-ventral patterning defects
Because germline clones of sec5E10 were lethal early in
oogenesis, we were unable to assess the subsequent roles of Sec5 during
cytoskeletal rearrangement and establishment of the anteroposterior and
dorsoventral axes. However, germline clones of the hypomorphic allele
E13 (truncated at position 361) are not lethal, and these females lay
eggs (Murthy et al.,
2003).
sec5E13 phenotypes in the germline were diverse, with
some egg chambers resembling those of the control
(Fig. 2D). Others possess
defects similar to those of sec5E10: phalloidin-marked
membranes are missing between cells, nurse cell nuclei appear to fall into the
oocyte where the membrane between them has broken down
(Fig. 2B) and ring canals clump
together (Fig. 2C). However,
all eggs laid by sec5E13 mothers show dorsoventral
patterning defects, similar to those caused by hypomorphic mutations in the
Gurken and EGF receptor signaling pathway
(Nilson and Schupbach, 1999;
Queenan et al., 1999
;
Roth et al., 1995
). Dorsal
appendages are either too closely spaced or are fused
(Fig. 2F).
|
In early egg chambers, Sec5 localizes to the membranes between cells in the germline, and to the side of the follicle cells that contacts the germline (Fig. 3A). Sec5 also appeared consistently enriched at the border between the posterior follicle cells and the egg chamber (Fig. 3B). In egg chambers where only the germline is mutant for sec5E13, this enrichment persists, indicating that it predominantly corresponds to the apical surface of the posterior follicle cells, particularly the two polar cells at the extreme posterior (Fig. 3C,D).
|
In late stage egg chambers, Sec5 protein alters its distribution within the oocyte membrane
At stage 6, Sec5 localization changes; although still enriched in the polar
cells and expressed at low levels ubiquitously, Sec5 concentrates at the
oocyte membrane (Fig. 3I). At
this time, the oocyte grows at a faster rate than the nurse cells and the
enrichment in the oocyte membrane may reflect the greater need for membrane
addition there. During stage 7, when the microtubule cytoskeleton reorients
and the nucleus moves to the dorsoanterior corner of the oocyte, Sec5 appears
enriched along this anterior rim, at the corners where the lateral and
anterior membranes of the oocyte meet (Fig.
3J), although still expressed all along the membrane. This pattern
continues through stage 8 (Fig.
3K). Finally, at stage 10, Sec5 is highly concentrated at the
anterolateral margins of the oocyte, with less detectable towards the
posterior end of the cell (Fig.
3M). To determine if this distribution was shared by other plasma
membrane-associated components of the membrane-trafficking apparatus, we
compared Sec5 labeling with that of the t-SNARE Syntaxin
(Burgess et al., 1997).
Syntaxin is present along the length of the oocyte membrane, including the
posterior region (Fig.
3N,O).
Because Sec5 becomes enriched at the anterior membrane of the oocyte at the
time when the microtubule cytoskeleton rearranges within the oocyte and
because the exocyst has been shown to associate with the cytoskeleton
(Vega and Hsu, 2001), we
tested if the localization of Sec5 was dependent on the cytoskeleton. In
oocytes from females treated with colcemid, a microtubule-depolymerizing drug,
Sec5 continued to be concentrated at the stage-appropriate domains of the
oocyte membrane, including the anterior corners at stage 10
(Fig. 3L).
The anterior trafficking of Gurken is disrupted in sec5E13 oocytes
The shift in Sec5 localization from the posterior of the oocyte to the
anterior parallels a shift in the directed secretion of Gurken
(Nilson and Schupbach, 1999).
Secreted at the posterior margin before stage 7, Gurken thereafter signals
from an anterior corner of the oocyte to adjacent follicle cells. Those cells
that receive the highest levels of Gurken repress the differentiation of the
dorsal lateral follicle cells, thus creating a space between two lateral
patches of cells that will form the appendages
(Morimoto et al., 1996
;
Neuman-Silberberg and Schupbach,
1994
). Because females with sec5E13 germlines
lay eggs with fused dorsal appendages (Fig.
2), we hypothesized a role for Sec5 in Gurken signaling.
In early stages, both wild type and sec5E13 germlines appropriately accumulated Gurken in the oocyte (Fig. 4A,D). After stage 7, however, Gurken was mislocalized in granules throughout the mutant oocytes (Fig. 4B,E). In stage 10 egg chambers, when Gurken is present at the dorsoanterior membrane of the oocyte in wild type (Fig. 4C), a substantial amount of Gurken is observed in granules scattered throughout the cytoplasm of sec5E13 oocytes. Much Gurken remains in the vicinity of the nucleus, but very little is present in the membrane (Fig. 4F,G). The cytoplasmic Gurken in sec5E13 oocytes is not coincident with a marker for the ER, Boca (data not shown), indicating that the block in the directed trafficking of Gurken is at a later step of the pathway.
|
In wild-type germlines, Yolkless is diffusely distributed until stage 8, whereupon, induced by an unknown signal, Yolkless translocates from the ooplasm to the cortex. At stage 7, Yolkless was detectable within both control and sec5E13 oocytes (Fig. 4J,K). At stage 8 in the mutant, however, the majority of the receptor did not go to the surface, and remained cytoplasmic through stage 10 (Fig. 4L,M). The mistrafficking of Yolkless, like the general disruption of membranes in the sec5 null allele, indicates that Sec5 is not only required for Gurken localization, but rather is of general significance for the membrane trafficking of many germline proteins.
The orientation of the microtubule cytoskeleton is undisturbed in sec5E13 oocytes
Although the Gurken and Yolkless mislocalizations were probably due to a
defect in membrane trafficking, these phenotypes might be secondary to a
defect in the concurrent reorganization of the oocyte, which includes the
reorientation of the microtubule cytoskeleton
(Theurkauf et al., 1992), the
movement of the oocyte nucleus to the anterior cortex of the oocyte, and the
localization of Gurken mRNA and protein near the nucleus.
To investigate this possibility, we examined the localization of several
proteins restricted to the posterior pole of the oocyte: Oskar, Par-1 and a
kinesin-ß-gal fusion (Clark et al.,
1994; Ephrussi and Lehmann,
1992
; Shulman et al.,
2000
). In both control and sec5E13 germlines,
all three proteins accumulate properly at the posterior pole in stage 8-10
oocytes (Fig. 5A-D). Dynein
Heavy Chain (Dhc), also localizes to the posterior end of late stage oocytes
(Brendza et al., 2002
). This
marker also was normal in the mutants, accumulating first in the oocytes of
early stage egg chambers (Fig.
5E,G) and after stage 8 at the posterior end of the oocyte
(Fig. 5F,H).
|
|
Defects in the membrane apposition of the nucleus in sec5E13 oocytes
Examining sec5E13 egg chambers, we noted that the
oocyte nucleus was sometimes mislocalized
(Fig. 5C;
Fig. 4F,G). The nucleus
invariably moved to the anterior, as in wild type, but was not closely
associated with the dorsoanterior plasma membrane. A three dimensional
composite image was assembled from individual z sections of stage 10
egg chambers and rotated to reveal the relationship of the nucleus to the
plasma membrane. This analysis confirmed that the nucleus was not always
adjacent to the dorsal membrane (Fig.
6I,J): eight out of 39 (21%) sec5E13 oocytes
had a mispositioned nucleus, but none of 40 wild-type oocytes.
Bicaudal-D (Bic-D) is a cytosolic protein that interacts with the
dynein-dynactin complex, and participates in the cortical anchoring of the
nucleus (Matanis et al., 2002;
Oh and Steward, 2001
;
Pare and Suter, 2000
;
Swan et al., 1999
). Bic-D
localized normally in sec5E13 oocytes throughout
oogenesis. In early stages, Bic-D was at the microtubule minus ends at the
oocyte posterior, and by stage 6 relocalized to the anterior rim, preceding
the arrival of the nucleus (Fig.
6K,L). Subsequently, Bic-D concentrated above the nucleus
(Fig. 6M,N). Even when the
oocyte nucleus was displaced from the dorsal cortex, Bic-D remained near the
nucleus, indicating that its nuclear association was not sufficient to attach
the nucleus to the dorsal cortex (Fig.
6O). Because we saw no alteration of the microtubule cytoskeleton
in sec5E13 germline clones, nor gross mislocalization of
Bic-D, the lack of a tight association of the nucleus with the membrane must
have other causes.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The trafficking of the Gurken protein provides at present the best example
of an identified membrane protein whose selective, Sec5-dependent localization
is crucial to proper development. The final deposition of Gurken is likely to
arise from a combination of mechanisms, including the transport of the oocyte
nucleus to an anterior corner, the nearby localization of Gurken mRNA, the
microtubule-dependent transport of Gurken protein to the cortex, and the
insertion of both pre-existing and newly synthesized Gurken into the plasma
membrane by vesicle fusion. The presence of displaced Gurken protein in the
posterior regions of the mutant ooplasm may be an indirect result of blocked
membrane fusion after which Gurken-containing vesicles may drift away from
their normal target. Gurken trafficking, however, also indicates that Sec5 and
the exocyst cannot be the only cues that direct vesicle fusion: Sec5 localizes
along the entire anterior lateral rim of the oocyte, but Gurken is inserted
only at that section adjacent to the nucleus. Furthermore, when the nucleus
and Gurken transcripts are mislocalized by cytoskeletal changes, some Gurken
signaling occurs ectopically, near the misplaced nucleus
(Ghiglione et al., 1999), and
away from the major concentration of Sec5
(Fig. 3L). Thus, the
localization of Sec5 should be viewed as one of several layers of likely
mechanisms for directing membrane proteins.
The phenotypes noted for sec5E13 oocytes were not due
to defects in the cytoskeleton, as the microtubules were properly oriented and
functional. Thus, sec5 mutations can affect membrane trafficking
without disrupting microtubule polarity or the localization of non-membrane
proteins. In this manner, the role of the exocyst in the oocyte appears to
parallel its role in yeast (Finger and
Novick, 1998; Novick et al.,
1980
; TerBush and Novick,
1995
). The oocyte findings are also consistent with proposed roles
in epithelial cells for the targeted delivery of proteins to basolateral
domains (Grindstaff et al.,
1998
).
Sec5 and E-cadherin in follicle cell signaling
A comparison of the sec5 phenotype in follicle cells to studies of
mammalian epithelia suggests both similarities and differences. In MDCK cells,
the exocyst has been shown to colocalize with E-cadherin at the tight
junctions between cells (Grindstaff et
al., 1998). In the ovaries, E-cadherin and Sec5 colocalize and
mutations in them similarly disrupt oocyte positioning and axis formation
(Godt and Tepass, 1998
). They
may therefore function in the same signaling pathway. Interestingly, however,
the signaling in the egg chamber is from the apical surface of the follicle
cells to the posterior surface of the oocyte. Heretofore, in polarized
epithelial cells, the exocyst has been reported to be required exclusively for
transport to the basolateral domain and to reside primarily at the tight
junction. The involvement of Sec5 in follicle cell to oocyte signaling is thus
surprising, as it implies a role for the exocyst in apical signaling. At
present, this role could either be a direct requirement in apical protein
insertion or an indirect role in establishing the adherens junctions and
thereby epithelial polarity.
Multiple membrane proteins depend on Sec5 for their traffic
In mediating the traffic of multiple membrane proteins, including both
Gurken and Yolkless, Sec5 is clearly in a distinct category from Cornichon and
Boca, proteins that act in the ER. These proteins are needed for the correct
transport of individual proteins and appear to act at earlier trafficking
steps. Gurken is retained inside the cell in cornichon mutants,
although vitellogenesis proceeds normally
(Roth et al., 1995). Boca,
however, is required for the trafficking of Yolkless and other LDL receptor
family proteins to the membrane, but does not influence Gurken traffic
(Culi and Mann, 2003
). These
highly specific deficits, which are likely to occur upon exiting from the ER
(Culi and Mann, 2003
;
Powers and Barlowe, 1998
), are
distinct from the more general disruption of traffic in sec5
mutants.
Many forms of membrane traffic to the cell surface now appear to depend on
the exocyst. In multicellular organisms, these include vesicles derived from
the trans-Golgi network (TGN) carrying newly synthesized proteins or mediating
neurite outgrowth (Grindstaff et al.,
1998; Vega and Hsu,
2001
; Murthy et al.,
2003
; Inoue et al.,
2003
; Sans et al.,
2003
). However, not all forms of exocytosis depend on the exocyst.
We have previously shown that the fusion of synaptic vesicles at nerve
terminals persists in sec5 mutants in which other trafficking events
are blocked (Murthy et al.,
2003
) and apical protein delivery in MDCK cells was resistant to a
block by antibodies to exocyst components
(Grindstaff et al., 1998
). The
essential differences between exocyst dependent and independent exocytotic
events remain unclear.
Is Sec5 required for earlier steps in protein transport?
In addition to an established role for the exocyst in targeting or fusion
at the plasma membrane (Grote et al.,
2000; Guo et al.,
1999
), there is also evidence to suggest a role at earlier stages
of protein traffic. The exocyst may associate with microtubules and a septin
protein, Nedd5, and thereby promote transport of post-Golgi vesicles to target
membranes (Vega and Hsu, 2001
;
Vega and Hsu, 2003
). Members
of the complex have been observed on perinuclear compartments in the cell
(Shin et al., 2000
;
Vega and Hsu, 2001
), and Sec6
and Sec8 are recruited to budding vesicles in the TGN, where antibodies
against these components interfere with the ability of cargo to exit the Golgi
(Yeaman et al., 2001
).
Recently, the exocyst component Sec10 has been found associated with
Sec61ß, a component of the ER translocon complex
(Lipschutz et al., 2003
).
Genetic interactions of sec61beta with members of the exocyst complex
have also been found (Lipschutz et al.,
2003
; Toikkanen et al.,
2003
). Indeed, mutations in sec61ß can cause
abnormal dorsal appendages very similar to those observed here for
sec5, probably owing to defects in Gurken translocation
(Valcarcel et al., 1999
).
In the present study, however, we have not detected exocyst functions at stages before exocytosis. Sec5 was concentrated only at the plasma membrane. Microtubule polarity and the polarized localization of cytosolic components were unaffected by the mutations. Perinuclear Gurken, a pool that is likely to represent protein in the ER and Golgi, was present in both wild-type and mutant stage 10 oocytes, but the mislocalized cytoplasmic granules of Gurken that characterize sec5E13 oocytes did not colocalize with the ER marker Boca. The early lethality of E10 clones, however, required that the analysis of Gurken and Yolkless trafficking be performed on the hypomorphic allele, E13. It is therefore possible that residual Sec5 function was sufficient for transport through the ER but insufficient at the plasma membrane.
The membrane association of the oocyte nucleus
Although some aspects of the sec5 phenotype can be ascribed to
defects in the transport of particular membrane proteins, such as Gurken and
Yolkless, others cannot, and these phenotypes may imply the existence of as
yet unidentified oocyte proteins. An example is the altered location of the
nucleus in late stage oocytes: whereas control nuclei were inevitably tightly
associated with the anterior membrane, in sec5E13 germline
clones, the nucleus was frequently displaced
(Fig. 6G-J). Members of the
dynein-dynactin complex are probably important for the association
(Swan et al., 1999). However,
Bic-D, a component of the dynein-dynactin complex, is anteriorly transported
and properly localized near the nucleus in sec5E13 clones.
We hypothesize, therefore, the existence of an as yet unidentified membrane
protein that tethers the oocyte nucleus to the cortex via the dynein-dynactin
complex. If the directed membrane traffic of this unidentified protein is
compromised in the sec5 mutants, the displacement of the oocyte
nucleus could be explained in a manner consistent with the other actions of
Sec5 in the oocyte.
Exocyst localization and the establishment of asymmetry
Owing to defects in directed traffic to the plasma membrane, aspects of the
anteroposterior axis and dorsoventral axis develop incorrectly in
sec5 mutant germlines. The requirement for Sec5 in directed membrane
traffic is consistent with previous studies in cells that use a polarized
secretory apparatus for cell growth and the transport of certain cargoes, such
as growing neurites (Hazuka et al.,
1999; Murthy et al.,
2003
; Vega and Hsu,
2001
), MDCK cells (Grindstaff
et al., 1998
; Lipschutz et
al., 2000
) and yeast (Finger
et al., 1998
).
The spatial correlation of membrane traffic with the position of the
exocyst raises the crucial question of how the exocyst acquires its
localization. This microtubule- and actin-independent mechanism remains
elusive (Fig. 3L)
(Finger et al., 1998). The
membrane receptor for the exocyst is not currently known, but in yeast Sec3p
may be the exocyst component closest to the membrane and its localization may
be controlled by Rho1p and Cdc42p (Drees
et al., 2001
; Zhang et al.,
2001
). In the Drosophila oocyte, the localization
mechanism must undergo developmental regulation to account for the shift in
localization that we have observed between stages 5 and 10. The mechanism that
targets the exocyst to the membrane and regulates the changes in its
localization is likely to be crucial to patterning and polarization in the
germline.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bolivar, J., Huynh, J. R., Lopez-Schier, H., Gonzalez, C., St
Johnston, D. and Gonzalez-Reyes, A. (2001). Centrosome
migration into the Drosophila oocyte is independent of BicD and egl, and of
the organisation of the microtubule cytoskeleton.
Development 128,1889
-1897.
Brendza, R. P., Serbus, L. R., Saxton, W. M. and Duffy, J. B. (2002). Posterior localization of dynein and dorsal-ventral axis formation depend on kinesin in Drosophila oocytes. Curr. Biol. 12,1541 -1545.[CrossRef][Medline]
Bretscher, M. S. (1996). Expression and
changing distribution of the human transferrin receptor in developing
Drosophila oocytes and embryos. J. Cell Sci.
109,3113
-3119.
Burack, M. A., Silverman, M. A. and Banker, G. (2000). The role of selective transport in neuronal protein sorting. Neuron 26,465 -472.[Medline]
Burgess, R. W., Deitcher, D. L. and Schwarz, T. L.
(1997). The synaptic protein syntaxin1 is required for
cellularization of Drosophila embryos. J. Cell Biol.
138,861
-875.
Chen, Y. A. and Scheller, R. H. (2001). SNARE-mediated membrane fusion. Nat. Rev. Mol. Cell Biol. 2,98 -106.[CrossRef][Medline]
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. Y. and Jan, Y. N. (1994). Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr. Biol. 4,289 -300.[Medline]
Clark, I. E., 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.
Craig, A. M., Wyborski, R. J. and Banker, G. (1995). Preferential addition of newly synthesized membrane protein at axonal growth cones. Nature 375,592 -594.[CrossRef][Medline]
Culi, J. and Mann, R. S. (2003). Boca, an endoplasmic reticulum protein required for wingless signaling and trafficking of LDL receptor family members in Drosophila. Cell 112,343 -354.[Medline]
Dollar, G., Struckhoff, E., Michaud, J. and Cohen, R. S. (2002). Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation. Development 129,517 -526.[Medline]
Drees, B. L., Sundin, B., Brazeau, E., Caviston, J. P., Chen, G.
C., Guo, W., Kozminski, K. G., Lau, M. W., Moskow, J. J., Tong, A. et al.
(2001). A protein interaction map for cell polarity development.
J. Cell Biol. 154,549
-571.
Ephrussi, A. and Lehmann, R. (1992). Induction of germ cell formation by oskar. Nature 358,387 -392.[CrossRef][Medline]
Ephrussi, A., Dickinson, L. K. and Lehmann, R. (1991). Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66, 37-50.[Medline]
Field, C. and Schekman, R. (1980). Localized
secretion of acid phosphatase reflects the pattern of cell surface growth in
Saccharomyces cerevisiae. J. Cell Biol.
86,123
-128.
Finger, F. P., Hughes, T. E. and Novick, P. (1998). Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 92,559 -571.[Medline]
Finger, F. P. and Novick, P. (1998). Spatial
regulation of exocytosis: lessons from yeast. J. Cell
Biol. 142,609
-612.
Ghiglione, C., Carraway, K. L., 3rd, Amundadottir, L. T., Boswell, R. E., Perrimon, N. and Duffy, J. B. (1999). The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis. Cell 96,847 -856.[Medline]
Godt, D. and Tepass, U. (1998). Drosophila oocyte localization is mediated by differential cadherin-based adhesion. Nature 395,387 -391.[CrossRef][Medline]
Gonzalez-Reyes, A. and St Johnston, D. (1998).
The Drosophila AP axis is polarised by the cadherin-mediated positioning of
the oocyte. Development
125,3635
-3644.
Grammont, M. and Irvine, K. D. (2002). Organizer activity of the polar cells during Drosophila oogenesis. Development 129,5131 -5140.[Medline]
Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Boulan, E., Scheller, R. H. and Nelson, W. J. (1998). Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 93,731 -740.[Medline]
Grote, E., Carr, C. M. and Novick, P. J.
(2000). Ordering the final events in yeast exocytosis.
J. Cell Biol. 151,439
-452.
Guo, W., Grant, A. and Novick, P. (1999).
Exo84p is an exocyst protein essential for secretion. J. Biol.
Chem. 274,23558
-23564.
Hawley, R. S. and Theurkauf, W. E. (1993). Requiem for distributive segregation: achiasmate segregation in Drosophila females. Trends Genet. 9, 310-317.[CrossRef][Medline]
Hazuka, C. D., Foletti, D. L., Hsu, S. C., Kee, Y., Hopf, F. W.
and Scheller, R. H. (1999). The sec6/8 complex is located at
neurite outgrowth and axonal synapse-assembly domains. J.
Neurosci. 19,1324
-1334.
Hsu, S. C., Ting, A. E., Hazuka, C. D., Davanger, S., Kenny, J. W., Kee, Y. and Scheller, R. H. (1996). The mammalian brain rsec6/8 complex. Neuron 17,1209 -1219.[Medline]
Inoue, M., Chang, L., Hwang, J., Chiang, S. H. and Saltiel, A. R. (2003). The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422,629 -633.[CrossRef][Medline]
Jahn, R. and Sudhof, T. C. (1999). Membrane fusion and exocytosis. Annu. Rev. Biochem. 68,863 -911.[CrossRef][Medline]
Jankovics, F., Sinka, R. and Erdelyi, M.
(2001). An interaction type of genetic screen reveals a role of
the Rab11 gene in oskar mRNA localization in the developing Drosophila
melanogaster oocyte. Genetics
158,1177
-1188.
King, R. C. (1970). The meiotic behavior of the Drosophila oocyte. Int. Rev. Cytol. 28,125 -168.[Medline]
Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H.,
Novick, P. and Mostov, K. E. (2000). Exocyst is involved in
cystogenesis and tubulogenesis and acts by modulating synthesis and delivery
of basolateral plasma membrane and secretory proteins. Mol. Biol.
Cell 11,4259
-4275.
Lipschutz, J. H., Lingappa, V. R. and Mostov, K. E.
(2003). The exocyst affects protein synthesis by acting on the
translocation machinery of the endoplasmic reticulum. J. Biol.
Chem. 278,20954
-20960.
Matanis, T., Akhmanova, A., Wulf, P., del Nery, E., Weide, T., Stepanova, T., Galjart, N., Grosveld, F., Goud, B., de Zeeuw, C. I. et al. (2002). Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex. Nat. Cell Biol. 4,986 -992.[CrossRef][Medline]
McGrail, M. and Hays, T. S. (1997). The
microtubule motor cytoplasmic dynein is required for spindle orientation
during germline cell divisions and oocyte differentiation in Drosophila.
Development 124,2409
-2419.
Morimoto, A. M., Jordan, K. C., Tietze, K., Britton, J. S.,
O'Neill, E. M. and Ruohola-Baker, H. (1996). Pointed, an ETS
domain transcription factor, negatively regulates the EGF receptor pathway in
Drosophila oogenesis. Development
122,3745
-3754.
Murthy, M., Garza, D., Scheller, R. H. and Schwarz, T. L. (2003). Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists. Neuron 37,433 -447.[Medline]
Neuman-Silberberg, F. S. and Schupbach, T.
(1994). Dorsoventral axis formation in Drosophila depends on the
correct dosage of the gene gurken. Development
120,2457
-2463.
Nilson, L. A. and Schupbach, T. (1999). EGF receptor signaling in Drosophila oogenesis. Curr. Top. Dev. Biol. 44,203 -243.[Medline]
Novick, P., Field, C. and Schekman, R. (1980). Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21,205 -215.[Medline]
Novick, P., Garrett, M. D., Brennwald, P., Lauring, A., Finger, F. P., Collins, R. and TerBush, D. R. (1995). Control of exocytosis in yeast. Cold Spring Harb. Symp. Quant. Biol. 60,171 -177.[Medline]
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]
Oh, J. and Steward, R. (2001). Bicaudal-D is essential for egg chamber formation and cytoskeletal organization in drosophila oogenesis. Dev. Biol. 232,91 -104.[CrossRef][Medline]
Pare, C. and Suter, B. (2000). Subcellular
localization of Bic-D::GFP is linked to an asymmetric oocyte nucleus.
J. Cell Sci. 113,2119
-2127.
Powers, J. and Barlowe, C. (1998). Transport of
axl2p depends on erv14p, an ER-vesicle protein related to the Drosophila
cornichon gene product. J. Cell Biol.
142,1209
-1222.
Queenan, A. M., Barcelo, G., van Buskirk, C. and Schupbach, T. (1999). The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila. Mech. Dev. 89, 35-42.[CrossRef][Medline]
Roth, S., Neuman-Silberberg, F. S., Barcelo, G. and Schupbach, T. (1995). cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila. Cell 81,967 -978.[Medline]
Ruden, D. M., Sollars, V., Wang, X., Mori, D., Alterman, M. and Lu, X. (2000). Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber. Dev. Biol. 218,314 -325.[CrossRef][Medline]
Sans, N., Prybylowski, K., Petralia, R. S., Chang, K., Wang, Y. X., Racca, C., Vicini, S. and Wenthold, R. J. (2003). NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat. Cell. Biol. 5, 493-495.[CrossRef][Medline]
Schonbaum, C. P., Perrino, J. J. and Mahowald, A. P.
(2000). Regulation of the vitellogenin receptor during Drosophila
melanogaster oogenesis. Mol Biol Cell
11,511
-521.
Shin, D. M., Zhao, X. S., Zeng, W., Mozhayeva, M. and Muallem,
S. (2000). The mammalian Sec6/8 complex interacts with Ca(2+)
signaling complexes and regulates their activity. J. Cell
Biol. 150,1101
-1112.
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 localization to the posterior pole. Cell 101,377 -388.[Medline]
Suter, B. and Steward, R. (1991). Requirement for phosphorylation and localization of the Bicaudal-D protein in Drosophila oocyte differentiation. Cell 67,917 -926.[Medline]
Swan, A., Nguyen, T. and Suter, B. (1999). Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning. Nat. Cell Biol. 1, 444-449.[CrossRef][Medline]
TerBush, D. R. and Novick, P. (1995). Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol. 130,299 -312.[Abstract]
Theurkauf, W. E. and Hazelrigg, T. I. (1998).
In vivo analyses of cytoplasmic transport and cytoskeletal organization during
Drosophila oogenesis: characterization of a multi-step anterior localization
pathway. Development
125,3655
-3666.
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.
Tkacz, J. S. and Lampen, J. O. (1972). Wall replication in saccharomyces species: use of fluorescein-conjugated concanavalin A to reveal the site of mannan insertion. J. Gen. Microbiol. 72,243 -247.[Medline]
Toikkanen, J. H., Miller, K. J., Soderlund, H., Jantti, J. and
Keranen, S. (2003). The beta subunit of the Sec61p
endoplasmic reticulum translocon interacts with the exocyst complex in
Saccharomyces cerevisiae. J. Biol. Chem.
278,20946
-20953.
Valcarcel, R., Weber, U., Jackson, D. B., Benes, V., Ansorge,
W., Bohmann, D. and Mlodzik, M. (1999). Sec61beta, a subunit
of the protein translocation channel, is required during Drosophila
development. J. Cell Sci.
112,4389
-4396.
van Eeden, F. and St Johnston, D. (1999). The polarisation of the anterior-posterior and dorsal-ventral axes during Drosophila oogenesis. Curr. Opin. Genet. Dev. 9, 396-404.[CrossRef][Medline]
Vega, I. E. and Hsu, S. C. (2001). The exocyst
complex associates with microtubules to mediate vesicle targeting and neurite
outgrowth. J. Neurosci.
21,3839
-3848.
Vega, I. E. and Hsu, S. C. (2003). The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. NeuroReport 14,31 -37.[CrossRef][Medline]
Vega-Salas, D. E., Salas, P. J. and Rodriguez-Boulan, E. (1988). Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells. J. Cell Biol. 107,1717 -1728.[Abstract]
Waddle, J. A., Karpova, T. S., Waterston, R. H. and Cooper, J. A. (1996). Movement of cortical actin patches in yeast. J. Cell Biol. 132,861 -870.[Abstract]
Wang, A. Z., Ojakian, G. K. and Nelson, W. J. (1990). Steps in the morphogenesis of a polarized epithelium. I. Uncoupling the roles of cell-cell and cell-substratum contact in establishing plasma membrane polarity in multicellular epithelial (MDCK) cysts. J. Cell Sci. 95,137 -151.[Abstract]
Yeaman, C., Grindstaff, K. K., Wright, J. R. and Nelson, W.
J. (2001). Sec6/8 complexes on trans-Golgi network and plasma
membrane regulate late stages of exocytosis in mammalian cells. J.
Cell Biol. 155,593
-604.
Zhang, X., Bi, E., Novick, P., Du, L., Kozminski, K. G.,
Lipschutz, J. H. and Guo, W. (2001). Cdc42 interacts with the
exocyst and regulates polarized secretion. J. Biol.
Chem. 276,46745
-46750.