In vivo membrane trafficking role for an insect N-ethylmaleimide-sensitive factor which is developmentally regulated in endocrine cells
1
Department of Cell Biology and Neuroscience, University of California,
Riverside, CA 92521, USA
2
Graduate Programs in Environmental Toxicology, Genetics, Biochemistry and
Molecular Biology, University of California, Riverside, CA 92521,
USA
* Author for correspondence at address 1 (e-mail: sarjeet.gill{at}ucr.edu )
Accepted 7 January 2002
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Summary |
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Key words: N-ethylmaleimide-sensitive factor, NSF, membrane trafficking, neuroendocrine secretion, development, Manduca sexta
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Introduction |
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NSF belongs to a superfamily of ATPases that are used in several cellular
contexts (Patel and Latterich,
1998). Mutations in the yeast NSF homologue, SEC18,
affect several steps in yeast biosynthetic and endocytotic pathways
(Graham and Emr, 1991
;
Hicke et al., 1997
;
Riezman, 1985
). Among these
fusion reactions, purified Sec18p has been shown to reconstitute endoplasmic
reticulum (ER)Golgi (Barlowe,
1997
), GolgiER (Spang
and Schekman, 1998
), and vacuolevacuole fusion
(Mayer et al., 1996
).
Functional conservation of NSF and Sec18p has been demonstrated by the ability
of yeast cytosol overexpressing Sec18p to mediate fusion in mammalian in
vitro systems employing N-ethylmaleimide (NEM)-treated membranes
(Wilson et al., 1989
;
Woodman et al., 1996
). Sec18p
is more resistant to NEM than to NSF
(Steel et al., 1999
) and,
given the presence of other NEM-sensitive factor(s) participating in several
fusion reactions (Goda and Pfeffer,
1991
; Rodriguez et al.,
1994
), these data show only indirectly that Sec18p is a functional
homologue of NSF. Thus far, purified Sec18p has been shown to function in a
heterologous environment only in permeabilized chromaffin cells
(Steel et al., 1999
).
Yeast cytosol overexpressing Sec18p could impart transport competence to
Chinese hamster ovary (CHO) Golgi membranes in vitro only when its
cofactor Sec17p was present. -SNAP (a cofactor for NSF) does not
substitute for Sec17p in these reactions
(Clary et al., 1990
;
Clary and Rothman, 1990
;
Wilson et al., 1989
).
Importantly, CHO NSF does not complement yeast SEC18 defects in
vivo (Griff et al.,
1992
), neither is it known to be functional in Sec18p-mediated
in vitro reactions. Cdc48p, the yeast homologue of p97 (related to
NSF by sequence), participates in ER membrane fusion. Although vertebrate
cytosol contains a potent yeast ER fusion activity, purified p97 is inactive
in these assays and p97 does not complement CDC48 defects in
vivo (Latterich et al.,
1995
). A simple interpretation for this lack of complementation is
that vertebrate NSF and p97 would require their corresponding SNAP(s)
(Clary et al., 1990
) and p47
(Kondo et al., 1997
) as
cofactors to be functional in yeast.
Information converging from different experimental approaches indicates
that homologues of participants in constitutive membrane trafficking might
play similar roles in regulated exocytosis of neurotransmitters
(Lin and Scheller, 2000) and
hormones (Burgoyne and Morgan,
1998
), but not enough is known about the molecular machinery
involved in hormone and neuroendocrine secretion in insect model systems
including Drosophila melanogaster. Our interest is in understanding
the role and function of homologues of transport proteins in endocrine
regulation of the developmental model, Manduca sexta. This system
offers several tangible advantages compared to Drosophila; in
particular, the neuroendocrine system and factors produced in it have been
studied to a greater degree than in any other developmental model
(Copenhaver and Truman,
1986b
).
We have begun an analysis of factors involved in neuroendocrine function of
M. sexta, borrowing from the expanding wealth of information in yeast
and mammalian cellfree systems. We have previously cloned the M.
sexta ortholog of NSF, MsNSF
(Pullikuth and Gill, 1999).
Here we characterize the in vivo function of MsNSF by using yeast as
a tractable model system. Our results show that an animal NSF could
genetically replace yeast Sec18p and does not require M. sexta
-SNAP-like protein(s) to be coexpressed to do so. Using an antibody
specifically raised against MsNSF, we identified a novel, developmentally
regulated role for MsNSF in neuroendocrine cells. Moreover, the selective
enrichment of MsNSF in corpora cardiaca (CC) and enteric-endocrine cells of
M. sexta suggests that MsNSF plays a preferential role in the
secretion of selective hormones, while secretion of others would either be
NSF-independent or might prefer as-yet-unidentified NSF homologue(s).
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Materials and methods |
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Yeast growth conditions
Strains were either grown in rich medium (1 % yeast extract, 2 % peptone)
supplemented with 2 % glucose (YPD) or 3 % galactose (YPG) or in minimal
synthetic medium [YNB: 0.17 % yeast nitrogen base without amino acids and
(NH4)2SO4] supplemented with 0.5 %
(NH4)2SO4 and amino acids, depending on the
selection required. Cells for metabolic labeling were grown in minimal medium
[containing 200 µmol l-1
(NH4)2SO4] with selective supplements lacking
methionine and uracil, from a saturated culture in rich medium.
Yeast expression construct
The coding sequence from MsNSF cDNA (approx. 5.5 kbp)
(Pullikuth and Gill, 1999),
was amplified with primers corresponding to the start and stop codon with
KpnI and NotI restriction sites, respectively. Upstream
(5'-TCCTGGTACCGGTCCATGTCTTCTATGCGTATGAAGGGAGGA-3') and
downstream primers
(5'-TTATGCGGCCGCTCATTCTTATTGAATAGTAGTGCCTAGATCTAG-3')
[underlined sequences correspond to coding region of the native open reading
frame (ORF)] were used in amplification with Long Template® polymerase
chain reaction (PCR) system (Roche Biochemicals). The amplified product was
cloned downstream from the GAL1 promoter in the yeast expression vector, pYES2
(URA3, Ampr, 2µm ori) (Invitrogen) to yield
the plasmid PGAL-MsNSF. This introduced a 55 bp spacer between the
GAL1 promoter and the initiator codon of MsNSF. The entire coding sequence of
MsNSF in PGAL-MsNSF was sequenced to confirm that no PCR errors
were incorporated. A BamHI/HindIII fragment containing the
entire reading frame and 5' untranslated region of the yeast
SEC18 (in pSEY8) was cloned into BamHI/HindIII
digested pBlueScript (SK+) to yield the construct pASH-SK18. A
BamHI/XhoI fragment from pASH-SK18 was cloned into pYES2 to
yield the plasmid pASH18GAL, (PGAL-SEC18). Yeast strains were
transformed by the lithium acetate method
(Ausubel et al., 1994
).
Immunoprecipitation of carboxypeptidase Y (CPY) and MsNSF from yeast
cells
Cells were grown overnight at 24 °C in YNB with 5 % glucose and
supplements, omitting uracil and methionine. Cells equivalent to an
A600 of about 50 were pelleted and grown at 24 °C in
induction medium (2 % galactose) for 2-8 h as indicated in the figure legends.
Prior to labeling, cells were preincubated for the times indicated in the
figure legends to impose a complete block of sec18-1. Metabolic
labeling was done with trans-35S (ICN Biochemicals) at 25
µCi (925 kBq)/1 A600 equivalent of cells. Labeling was
stopped by the addition of a chase mixture containing 20 mmoll-1
each of methionine, cysteine and (NH4)2SO4.
Chase portions (5-10 A600 units) were precipitated with 10
% trichloroacetic acid (TCA) on ice. TCA precipitates were pelleted and washed
twice with acetone (prechilled at -20 °C) and dried under vacuum.
Acetonedried pellets were disrupted with glass beads and immunoprecipitation
carried out as previously described
(Gaynor and Emr, 1997) using
Protein-A sepharose CL4B to sediment the immunocomplexes. After the final
wash, the beads were dried under vacuum, resuspended in SDS-PAGE sample buffer
and analyzed by SDS-PAGE. Fluorography was performed with Entensify®
solutions (DuPont).
Labeling cells for media proteins
About 10 A600 equivalents of cells grown as above were
concentrated and resuspended in fresh medium and preincubated for 15 min at 37
°C. Bovine serum albumin (500 µg ml-1) and
2-macroglobulin (0.05 units ml-1, Roche Biochemicals) were
added prior to preincubation. Cells were labeled and chased as above. The
chase was stopped by the addition of NaN3-NaF on ice at a final
concentration of 20 mmoll-1 each. The culture medium was separated
from cells by centrifugation at 14 000 g for 5 min. Cell
pellets and media were precipitated separately with 10 % TCA on ice. TCA
precipitates were washed with acetone and dried as described above. Pellets
were resuspended by sonication in 50 µl of TETx (10 mmoll-1
Tris-HCl, pH 7.5, 1 mmoll-1 EDTA, 0.5 % Triton X-100), an equal
volume of SDS-PAGE sample buffer (with 5 % 2-mercaptoethanol) was added, and
the samples were heated at 85 °C for 15 min. Denatured samples were
clarified at 12 000 g for 5 min and equal
A600 equivalents of the supernatants analyzed by SDS-PAGE.
Acetone-washed cell pellets were lysed with glass beads and divided into two
portions. One portion was immunoprecipitated with
-MsNSF and the other
with
-CPY to check for any block of CPY in sec18-1
mutants.
Analysis of HSP150 trafficking
Cells were grown as for labeling medium proteins but in rich medium, YPG (3
% galactose). Equal portions were washed twice in water, once in fresh medium
and resuspended in prewarmed medium (37 °C) and incubated at 37 °C for
30 min. Cycloheximide was added to a final concentration of 20 µg
ml-1 (from a stock of 2 mg ml-1 in 95 % ethanol).
Samples were removed just before (0 min chase) or 30 min after the addition of
cycloheximide (see Fig. 3C
legend). Cells and media were transferred to 20 mmoll-1
NaN3-NaF on ice and separated by centrifugation (14 000
g, 5 min). Cell and media proteins were TCA-precipitated,
acetone-washed and air-dried. Precipitates were separated by SDS-PAGE,
immunoblotted with anti-HSP150 antibody (1:5000) and detected with anti-rabbit
horseradish peroxidase (HRP) antibody (diluted 1:2500) using ECL (Amersham
Pharmacia).
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Antibodies to insect neuropeptides and hormones
Affinity-purified -MsNSF antibodies have been described
(Pullikuth and Gill, 1999
).
Antibodies to insect hormones and peptides were generous gifts from the
following sources: diuretic hormone and leucokinin (J. Veenstra, University of
Bordeaux, France), eclosion hormone, C terminal (Drs T. G. Kingan and M. E.
Adams, University of California, Riverside, CA, USA), ecdysis triggering
hormone (Drs D. Zitnan and M. E. Adams), proctolin and FMRFamide (T. G.
Kingan), bombyxin (D. Zitnan), M. sexta prothoracicotropic hormone,
PTTH (Dr W. Bollenbacher, University of North Carolina, NC, USA), small
cardioactive peptide B, SCPB (Dr D. Willows, University of
Washington, WA, USA), silkmoth period and PTTH (Drs I. Sauman, S. Reppert and
T. Gotter) and Drosophila synaptotagmin (Dr Hugo Bellen, Baylor
College of Medicine, Texas, USA). Monoclonal antibody (HPC-1) to syntaxin was
purchased from Sigma (St Louis, MO, USA).
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Results |
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Rescue of sec18-1 temperature-sensitive growth defect by heterologous
expression of MsNSF
Drosophila has two NSF homologues
(Boulianne and Trimble, 1995;
Ordway et al., 1994
;
Pallanck et al., 1995b
);
however, neither is known to be involved in intra-Golgi transport in
vitro or in vivo, much less to be functional in yeast. The
mutant allele sec18-1 exhibits temperature-sensitive growth
inhibition at
28 °C, whereas its growth is similar to wild type at 24
°C (Fig. 1A). To test if
MsNSF possesses Sec18p-like activity, MsNSF was expressed under the regulation
of the GAL1 promoter, which could be induced to high levels with galactose
(Table 2). Sec18-1
cells containing PGAL-MsNSF or SEC18 gene with its native
5' region, or PGAL-SEC18, were spotted on
galactose-containing medium and grown at permissive and non-permissive
temperatures (Fig. 1A,B). The
temperature-sensitive growth was rescued by MsNSF expression in
sec18-1 cells, which was comparable to wild-type or
SEC18-carrying cells at 30 °C
(Fig. 1A). Without prior
induction of MsNSF, complementation was only modest at 37 °C
(Fig. 1A).
|
Glucose suppresses GAL1 promoter activity. Raffinose neither suppresses nor induces GAL1 promoter, suggesting that the GAL1 promoter is constitutively active when raffinose alone is provided as carbon source. Cells were grown as above and spotted on medium containing different carbon sources. As shown in Fig. 1B, sec18-1 failed to grow at 32 °C, but this was alleviated by MsNSF expression when induced with galactose. This rescue was completely suppressed by glucose and raffinose, which indicates that the growth phenotype was a direct effect of MsNSF expression. Under constitutive levels of expression with raffinose, MsNSF failed to rescue the temperature-sensitive phenotype (Fig. 1B), strongly suggesting that the rescue was dose-dependent. It is noteworthy that PGAL-SEC18 could not be completely suppressed by glucose or raffinose, suggesting that it is constitutively expressed due to the presence of 5'-region of SEC18 in the construct. Constitutive levels of Sec18p are thus sufficient to rescue temperature-sensitive growth in sec18-1 mutants.
Several of the sec mutants accumulate intra-cellular structures
and lyse after prolonged exposure to non-permissive temperatures
(Novick et al., 1980;
Riezman, 1985
). Cells were
incubated at 37 °C for 8 days, returned to 22 °C and incubated for 4
days. MsNSF-containing cells remained viable and were capable of growth after
this prolonged exposure to restrictive temperature, while sec18-1
cells as expected were unable to revive growth
(Fig. 1C). These data clearly
demonstrate that an animal NSF is capable of rescuing the
temperature-sensitive phenotype when expressed in sec18 mutant, and
does not require the activity of animal SNAPs to do so.
To obtain biochemical support for the above data, cells grown at various
induction conditions were analyzed by immunoblotting lysates with
-MsNSF and
-Sec18p antibodies
(Table 2). Induction time and
carbon source affected the level of expression of MsNSF. Twice as much MsNSF
was expressed after induction for 4 h in galactose, compared to Sec18p
expressed from its native 5' region; this level only marginally rescued
the temperature-sensitive defect at 37 °C. However, rescue was complete up
to 32 °C (Fig. 1). MsNSF
levels needed to be eight- to ninefold higher than native Sec18p for complete
complementation. Due to large variations between experiments it was not
possible to conclusively identify the minimal level of MsNSF required for
rescue, and the levels reported here might be overestimated.
Rescue of transport defect by expression of MsNSF
In yeast, SEC18 is an early acting gene, and defects in it cause
secretory proteins to arrest in the ER-modified forms when shifted to
restrictive temperatures. Additional defects in sec18-1 mutant were
detected in intra-Golgi and Golgi-plasma membrane routes of transport
(Graham and Emr, 1991). The
data shown above clearly indicate the feasibility of using yeast as a model
system to understand the in vivo role(s) of proteins from
heterologous systems where assays to understand such roles are not available.
To analyze the role of MsNSF in intracellular protein transport, we chose
three well-characterized markers, carboxypeptidase Y (CPY), alpha factor
(
-F) pheromone and heat shock protein 150 (HSP150).
MsNSF restores CPY transport to the vacuole in the sec18-1
mutant
CPY is a 61-kDa subunit enzyme of which 10kDa is contributed by four
N-linked oligosaccharide modifications. In wild-type cells, CPY
targeted to the ER is modified by the addition of core oligosaccharides (p1
form, 67kDa), and is further modified in the Golgi (p2 form, 69kDa). The p2
form is sorted to the vacuole where it is proteolytically processed to yield
the mature form of the enzyme (mCPY, 61 kDa)
(Hasilik and Tanner, 1978;
Stevens et al., 1982
).
Mutations in SEC18 affect the transit of CPY to the distal Golgi
compartment (Graham and Emr,
1991
). If MsNSF could replace the defective Sec18p in
vivo then the ER-arrested form (p1) should be matured to mCPY under
restrictive conditions. Fig. 2
shows the comparative maturation of CPY in sec18-1, MsNSF and
SEC18 cells. After induction for 2h, when cells were labeled after
incubation at 33°C for 30 min, sec18-1 cells accumulated p1 CPY,
which failed to mature during the chase period
(Fig. 2A, lanes 1-3). This
transport block was rescued by the expression of MsNSF, which yielded the
mature vacuolar form, mCPY (lanes 10-12). MsNSF mediated rapid vacuolar
transport since 42% mCPY was recovered at 0 min of chase (lane 10). The above
data was quantitated from three experiments by densitometry of digitized
images. In MsNSF-, SEC18- and
PGAL-SEC18-expressing cells, mCPY maturation, after a 30
min chase, was approx. 78 %>95 % and 84%, respectively
(Fig. 2A). Since expression
levels under longer induction showed phenotypic rescue, cells were induced for
6 h before temperature shifts and analyzed for CPY maturation
(Fig. 2B,C). Consistent with
the phenotypic data (Table 2,
Fig. 1), higher levels of MsNSF
expression facilitated mCPY formation, but was kinetically slower than wild
type (Fig. 2, compare lane at
30 min in B with corresponding lane in C). From these results we conclude that
MsNSF expression could substitute for a mutant Sec18p in vivo in
mediating intracellular trafficking of CPY. Functional substitution by MsNSF
does not require any other fusion-promoting factors such as Sec17p or Rab
homologues from M. sexta to exert its action in yeast. This is the
first demonstration of an in vivo expression-dependent function for
an animal NSF in intra-Golgi transport.
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Intra-Golgi transport of yeast pro-alpha-factor is restored by MsNSF
in sec18 mutants
In sec18 cells, the mating pheromone -F is predominantly in
the ER-modified form under non-permissive conditions. Transfer to the Golgi
results in the addition of complex sugars in a compartment-specific manner,
producing the hyperglycosylated form. In a distal Kex2p-containing compartment
the prohormone is processed to the mature 13-amino-acid-residue active
pheromone that is secreted into the medium. The addition of different sugars
at distinct levels of Golgi organisation results in distinguishable forms of
the prohormone that can be resolved by their migration on SDSPAGE. The
nature of sugar modification indicates the level of Golgi organisation that
the protein has reached. In sec18-1 cells at 33 °C, only the core
glycosylated ER form predominated, which failed to mature into the Golgi forms
during the 30 min chase period at non-permissive temperature
(Fig. 3A, lanes 1,2). The
complete lack of
-1,6 and
-1,3 modified Golgi forms in the
immunoprecipitates strongly suggested that, under our conditions, the core
glycosylated prohormone was never delivered to the Golgi. In contrast, in
MsNSF-expressing cells, even at 0 min chase, the predominant forms were
Golgi-modified, corresponding to the sizes of both
-1,6 and
-1,3
mannose added forms (G; Fig.
3A, lane 5). These results suggested that the functionality
imparted by active MsNSF is sufficient to provide rapid transport of
-factor through the compartment. After a chase for 30 min, cells were
shifted to permissive temperature (24°C) to allow for resumption of
transport of ER-locked forms (lanes 3,4 and 7,8) and to demonstrate that
-factor in sec18-1 cells can acquire Golgi-specific
modifications as expected for permissive conditions. It should be noted that
MsNSF expression resulted in a rapid disappearance of Golgi-modified form,
presumably by secreting the mature protein into the medium
(Fig. 3A, lane 7; see legend).
Together the data (Figs 2,
3A) suggest that MsNSF
alleviates a Sec18p defect in vivo for two well-characterized
secretory markers in intra-Golgi transport.
Golgi to plasma membrane and exocytosis
Consistent with the NSF interaction with neuronal SNAREs
(Söllner et al., 1993b),
Graham and Emr (1991
) showed
that Sec18p acts in the exocytotic pathway from post-Golgi to plasma membrane.
However, purified Sec18p has not been shown to be required for this step. We
used a secretion assay (Gaynor and Emr,
1997
) to determine if MsNSF could functionally restore the late
stage in exocytosis. This assay does not place bias on any particular protein
but qualitatively assesses whether proteins in a size range are secreted into
the medium after imposing a temperature block prior and during pulse-chase
protocols. Cells were grown in induction medium and subjected to restrictive
conditions for 30 min at 37°C (Fig.
3B). After being subjected to a pulse-chase protocol, equal
portions of labeled cultures were separated into medium and cell fractions,
and analyzed by SDS-PAGE and fluorography.
Medium proteins were absent from sec18-1 mutants
(Fig. 3B, lane 2). At least
seven protein bands (arrows, Fig.
3B) were apparent in sec18-1 complemented with MsNSF
(Fig. 3B, lane 4), similar to
SEC18-carrying cells (Fig.
3B, lane 3). Among the predominant proteins whose secretion was
blocked by SEC18 mutation, the protein of approx. 150kDa
(Fig. 3B, lanes 1,3,4)
corresponds to the well-characterized heat shock protein HSP150
(Russo et al., 1992).
HSP150 is induced approximately sevenfold under heat stress and is
extensively O-glycosylated (Russo
et al., 1992). Functional Sec18p is required for the secretion of
HSP150 since in sec18-1 it is predominantly found in the ER form
under restrictive conditions (Gaynor and
Emr, 1997
). Cells were incubated at 37°C for 30 min, washed in
prewarmed (37°C) medium and incubated for 30 min with cycloheximide
(Fig. 3C). Portions were
removed prior to addition of cycloheximide (0 min chase, odd-numbered lanes).
Cells were collected at 37°C, washed in prewarmed medium twice, and
incubation continued for 30 min with cycloheximide
(Fig. 3C, even-numbered lanes).
Medium proteins were separated by electrophoresis and analyzed by
immunoblotting with HSP150 antibody. As expected in wild type (WT), HSP150 was
efficiently secreted into the medium (Fig.
3C, lanes 1,2), whereas no detectable HSP150 was found in the
medium from sec18-1 cells (Fig.
3C, lanes 3,4). Expression of MsNSF or SEC18 rescued this
block of HSP150 (Fig. 3C, lanes
5-10). Sec18p has not been localized to fusion complexes formed during
exocytosis, i.e. the exocyst complex, nor has purified Sec18p been shown to
reconstitute this step in vitro. However, in sec18-1
mutants, components of this complex are localized to discrete sites in the
plasma membrane, indicating a potential role for Sec18p in mediating
exocytosis from the exocyst complex formed at the plasma membrane
(Carr et al., 1999
). The fact
that MsNSF could relieve this late Golgi transport step suggests that MsNSF is
indeed functional in post-Golgi trafficking, and could replace a mutant Sec18p
defect precisely at that point of the exocytotic pathway.
Stage-, tissue- and cell-type-specific expression of M. sexta
NSF
The level and pattern of MsNSF protein expression were analyzed with
antibodies against the D1 domain of MsNSF. These preparations specifically
recognized a 83kDa protein in immunoblots, consistent with the estimated size
of MsNSF. MsNSF expression was highest in the central nervous system and the
brain of larval and pupal stages of M. sexta
(Fig. 4A), extending our
previous observation (Pullikuth and Gill,
1999). Preimmune sera, or sera depleted by preincubating with the
D1 domain of MsNSF, completely abolished the signal in immunoblots (not
shown). Affinity-purified antibodies specifically immunoprecipitated
in-vitro-labeled MsNSF (Pullikuth
and Gill, 1999
) and metabolically labeled MsNSF from cells
carrying the MsNSF expression vector, but not from isogenic cells expressing
Sec18p from a multicopy vector (pSEY8) or from an inducible expression vector
(PGAL) (Figs 2,
3B).
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M. sexta is an excellent model to study neuroendocrine
determinants in animal development as it has already been used as a system to
identify and isolate several hormones and peptides involved in metamorphosis.
The ontogeny and physiological roles of neuroendocrine cells in this system
have been resolved to a far greater extent than in any other insect species
(Copenhaver and Truman,
1986a,b
).
Although two isoforms of NSFs (dNSF1 and 2) have been isolated in
Drosophila, their expression pattern at the protein level is not well
understood. dNSF1 mRNA is expressed at higher levels in head and embryonic CNS
(Ordway et al., 1994
), whereas
dNSF2 is ubiquitously expressed, suggesting a more generalized role in
constitutive protein trafficking (Boulianne
and Trimble, 1995
; Golby et
al., 2001
; Pallanck et al.,
1995b
). Thus far, NSF protein has not been localized in endocrine
organs in the brain. We focused on three aspects for further characterization.
First, the enteric nervous system (ENS) was used as a model to understand the
non-neuronal distribution. Second, we analyzed the anterior protocerebral
neurosecretory complex during all developmental stages. Finally, we
characterized a novel NSF reactivity in a specific set of neurosecretory cells
that are regulated in development.
MsNSF in enteric nervous system (ENS)
M. sexta ENS consists of 80-100 neurons clustered in the enteric
plexus in the anterior midgut (Fig.
4B). These neurons send axonal projections along the entire length
of the organ (B) and innervate the proctodeal nervous system, terminating in
varicosities indicative of hormone release sites
(Truman, 1992). Apart from the
ENS neurons, which modulate muscle activities of midgut, a slew of
endocrine/paracrine cells (EPC) lines the entire length of midgut. These cells
are peptidergic in nature and contain molecules antigenically similar to
vertebrate neuropeptides and hormone. Further, the midgut is a rich source of
ecdysteroids, which dictate the fate of the developing insect by rising and
falling titers. ENS neurons and EPC could be marked by FMRFamide reactivities
that stain a variety of peptides with FMRF epitopes
(Fig. 4B,C)
(Zitnan et al., 1993
).
Whole-mount immunohistochemistry was performed on isolated midguts from
neonate larvae. Two distinct patterns of NSF reactivities were evident. A
diffused staining pattern, apparent only with
-MsNSF antibodies, which
we term as Type I NSF immunoreactivity (NSF-IR), was evident. This signal was
specific since negative control experiments done in parallel produced little
staining (not shown). Type I NSF-IR reflects the presence of MsNSF in most
cells, consistent with its constitutive role(s). On the other hand, a pattern
of enriched NSF-IR, which we term eNSF-IR, was restricted to perhaps six rows
in the median midgut (Fig. 4D).
FMRFamide reactivities were present in approximately 12 rows of EPC along the
entire length of midgut (Fig.
4C). eNSF-IR was excluded from ENS neurons and axons, and from EPC
of anterior and posterior midgut.
EPCs consist of two predominant types of cells, closed and open. Larval midgut consisted of one (Fig. 4F) or two (Fig. 4F, inset) eNSF-IR cells per invagination exclusively restricted to the closed cell type. We call them pediculate EPCs, since stalked processes project into the lumen of the midgut. No eNSF-IR was found in open EPCs. The eNSF-IR EPC cell body is exposed to hemolymph and thus strategically poised to relay hormonal information from the hemolymph to the midgut lumen. The spectra and identity of hormones produced in each type of EPC are not known, although many react to antibodies against vertebrate neuropeptides and hormones, and are thus thought to contain antigenically similar factors.
The midgut is composed primarily of two cell types, columnar cells and secretory goblet cells. An intermediate staining pattern, termed Type III, with a median midgut bias was found mostly restricted to the basolateral domain of goblet cells (Fig. 4E). Columnar cell staining was unremarkable. The localization pattern of MsNSF in the basolateral domain is consistent with studies performed in vitro where vertebrate NSF was shown to be required for transport from the TGN to the basolateral membrane. Diminished or lack of staining in most other cells points to the possibility that NSF-independent mechanisms, or other unidentified NSF isoforms, might be involved in trafficking in a majority of midgut cells. Exclusion of NSF-IR in enteric neurons and axons bolsters the claim that multiple MsNSF isoforms or functionally similar molecules would govern release processes in this important group of neurons, modulating various aspects of feeding, digestion and ecdysis. The selective expression of NSF is likely to exist in other species, as Drosophila midgut and gastric ceacae exhibited regional and cell-type-specific NSF enrichments with the same antibodies (A. K. P., M. Filippova and S. S. G., unpublished observations). In the Malpighian tubules, most NSF-IR was found restricted to the plasma membrane (Fig. 4G,I).
NSF reactivity in optic and antennal lobes of M. sexta
In the optic lobe most photoreceptors exhibited NSF-IR, largely restricted
to cytoplasmic regions (Fig.
4M). Three granular structures of the optic lobe, namely lobula,
medulla and lamina, stained for MsNSF (Fig.
4K,L), indicating a role for MsNSF in visual perception and
integration of sensory information in M. sexta. No specific groups of
cells of the optic lobe showed eNSF-IR. Distinct groups of cells containing
neuropeptides and neurotransmitters have been localized in the optic lobe;
however, the lack of specific enrichment in any of these cells suggests that
MsNSF is expressed to similar extent in most types of peptidergic and
neurotransmitter-containing cells of the optic lobe. In the antennal lobe,
glomeruli (g) stain for MsNSF (Fig.
4H) where sensory inputs are integrated. Afferent nerves from
glomeruli innervate the mushroom body, a critical structure implicated in
learning and memory in insects.
NSF-IR in the neurosecretory complexes of M. sexta brain
Components of NSF-mediated complex have been predicted to regulate
exocytotic events, including hormonal secretion. However, to date, there is no
direct evidence for the presence of NSF in insect neurosecretory cells or
sites of hormone release. The protocerebral neurosecretory complex of M.
sexta consists of several paired cells with distinct content catalogue
and defined axonal ramifications (Copenhaver and Truman,
1986a,b
;
Zitnan et al., 1995a
).
Whole-mount immunohistochemistry was performed on brains from staged and sexed
animals. The expression pattern of MsNSF was unremarkable in larval brain.
Surprisingly, beginning with day 3 after pupal ecdysis, four cells in the
anterior protocerebrum showed eNSF-IR (Fig.
5C) apart from the specific diffused Type I-IR found in all
regions (Fig. 5B). eNSF-IR in
these two pairs was sustained throughout later development and persisted after
adult emergence (Fig. 5C-E).
eNSF-IR in these cells was not sexually dimorphic (not shown), thus was
unlikely to be involved in sex-specific traits. To rule out the possibility
that the size of tissues in whole mounts might mask other reactivities,
paraffin sections of pharate adult were examined with the same antibody.
Confirming our results, only four cells expressed the eNSF-IR phenotype
(Fig. 5G, arrow; only one from
each pair in the plane of section shown). eNSF-IR was not due to problems of
antibody penetration since
-FMRFamide stained several groups of cells
and axons at all levels (Fig.
5F). Synaptotagmin and syntaxin antibodies stained all cells in
the protocerebral complex equally, suggesting that these important components
in membrane fusion are not preferentially expressed in these cells (data not
shown).
|
Axonal processes from these eNSF-IR cells were weakly detected after 7 days
of pupal ecdysis, gradually increasing in staining intensity toward adult
development (Fig. 6F). Brain
eNSF-IR is curiously poised within a cluster of cells constituting the major
neurosecretory atrium in protocerebrum. Hormones involved in homeostasis,
cellular differentiation, egg development, vitellogenesis and eclosion have
all been localized to specific sets of cells within this complex. Peptides and
hormones produced from this complex are released from either of the two major
neurohemal organs, corpora cardiaca (CC) and corpora allata (CA), both
posterior glandular structures connected to the brain through nervi corpora
cardiaca (NCC). Despite both CC and CA being principal sites of
peptide/hormone release, each is involved in releasing a subset of hormones
during development. Given the putative role of NSF in mediating
neurotransmitter release, we examined MsNSF expression in peptide/hormone
release structures in M. sexta. CCCA complexes from ecdysing
pupa to adult insects were analyzed by whole-mount immunohistochemistry with
affinity-purified -MsNSF. Remarkably, only CC was found to produce
eNSF-IR with distinct varicosities characteristic of hormone release sites
(Fig. 5H,I). However, this
pattern of eNSF-IR in CC was also present in larval stages (data not shown),
albeit less intense. The preferential enrichment of NSF in one of the two
major sites for hormone/peptide release suggests that it is involved in
regulating the release of only a subset of factors produced in the brain. No
change in eNSF-IR in CC was detected between larvallarval,
larvalpupal, and pupaladult ecdyses, ruling out the possibility
that MsNSF is directly associated with ecdysis and eclosion.
|
To understand if eNSF-IR in M. sexta brain colocalized with
distinct neuropeptides, immunostaining was performed with antibodies against
eclosion hormone (EH) (Copenhaver and
Truman, 1986a), PTTH
(Westbrook et al., 1993
),
diuretic hormone (DH), leukokinin
(Veenstra and Hagedorn, 1991
),
period (Sauman et al., 1996
)
and bombyxin (Zitnan et al.,
1990
,
1995b
). Bombyxin, an
insulin-related molecule that primes the prothoracic glands to secrete the
ecdysis hormone, ecdysteroid, is produced in four pairs of cells. Since the
location of bombyxin cells was similar to eNSF-IR cells, double immunostaining
with
-MsNSF and
-bombyxin was performed. Signals were detected
with anti-mouse-cy2 conjugated secondary antibody to visualize bombyxin
reactivity (Fig. 6A, green),
while anti-rabbit-cy3 conjugated antibody detected MsNSF-staining cells
(Fig. 6A, red). These eNSF-IR
cells did not coincide with any of the bombyxin-reactive cells as apparent
from the lack of colocalization. Another candidate set of cells is those
producing DH, which localize to the dorsal protocerebrum in larval stages.
During pupaladult development, DH production is shut off in these cells
and production is shifted to a cluster of 8-10 smaller cells in early pupa and
80-100 cells in late pupa/adult (Fig.
6B), all probably arising from a common neuroblast in later stages
(Veenstra and Hagedorn, 1991
).
The presence of these cells in later stages is controversial, since DH
reactivity is the only known criterion for their identification. During adult
metamorphosis these cells are thought to be lost or atrophied. However,
-MsNSF antibodies detect two pairs lateral to the cluster of 80-100
cells staining for DH (Fig.
6C). The sheer size difference and localization rules out any
subset of DH cells to express eNSF-IR. However, it should be noted that these
are the same cells that express DH in larva.
Eclosion hormone (EH) is one of the major determinants of ecdysis in M.
sexta and directs its action through a cascade of hormonal signaling. EH
in larva is produced by two pairs of ventro-medial (VM) cells
(Fig. 6D). VM cells undergo
migration concomitant with the gross rearrangement of the brain during pupal
and adult development. Fusion of subesophageal ganglion with the brain
coincides with VM cells being ventrally positioned in the anterior
protocerebrum of late pupa/adult brain
(Fig. 6E). EH in late stages is
thought to be released primarily from CC-CA complex, as distinct from the
proctodeal system in larva. To rule out the possibility that eNSF-IR cells
were EH cells, cells stained for -EH
(Fig. 6E) and eNSF-IR
(Fig. 6F) were examined both
from dorsal and ventral aspects. Axons from eNSF-IR cells project ventrally
and run median to VM axons and project to CC through NCC I and II similar to
DH-IR in larva (Fig. 6F). This
allowed unequivocal assignment of eNSF-IR cells to Type IIa4 cells.
These data rule out most of the known hormones and peptides as candidates for
colocalization with eNSR-IR, and thus it constitutes a novel reactivity.
![]() |
Discussion |
---|
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---|
In vivo complementation by MsNSF in yeast occurs without its
cofactor SNAP(s) from a homologous source. The higher expression levels
required for functional complementation may be due to differences in
properties of MsNSF such as its ATPase activity, affinity for Sec17p, or
requirement for SNAP (-like) cofactors from an identical source and
recognition of SNARE complexes in their right context and from the correct
species. Vertebrate p97 (yeast Cdc48p) which, like NSF, has been implicated in
fragment assembly of mitotic Golgi
(Rabouille et al., 1995) is
not functional in a yeast Cdc48p assay for ER membrane fusion; however, crude
lysate from Xenopus possesses greater Cdc48p activity in
vitro (Latterich et al.,
1995
). A reason for lack of complementation by vertebrate NSF and
p97 in yeast SEC18 and CDC48 mutants might be that these
proteins require their respective cofactors
-SNAP and p47 to be active
in heterologous environments.
Each subunit of NSF hexamer can be divided into distinct domains (for a
review, see May et al., 2001).
The D1 domain provides the major ATPase activity in disassembling SNARE
complexes (Matveeva et al.,
1997
; Nagiec et al.,
1995
; Steel and Morgan,
1998
). ATP binding to D2 is required for hexamerization of NSF but
not for SNARE disassembly (May et al.,
2001
). The temperature-sensitive mutation of SEC18
(89G
D) occurs in the N-domain implicated in interacting
with SNAREs (A. Morgan; cited by May et
al., 1999
). Sequence divergence of this region among cloned NSFs
might reflect its structural requirement for interacting with species-specific
SNAP-SNAREs. By this criterion, one would expect MsNSF to have lower affinity
for yeast SNAREs than Sec18p. Since the N-domain contains a critical pocket
proposed to interact with SNAP and, in turn, with SNARE complex, subtle
structural differences in this region would underlie the ability to
discriminate the substrate from the right species. Another possibility is that
MsNSF might be interacting with the mutant form of Sec18p in vivo to
form a less functional heterooligomer, rather than all subunits being
contributed by MsNSF to form a fully functional homo-hexamer. In
vitro experiments suggest that this is an unlikely situation since NSF
oligomer containing even a single mutant subunit is completely defective in
fusion reactions (Whiteheart et al.,
1994
).
NSF assembles into a 20 S complex containing SNAREs and SNAP
(Wilson et al., 1992). The 20
S complex contains three copies of SNAP bound to one hexameric NSF
(Wimmer et al., 2001
).
Neuronal (Hayashi et al.,
1995
; Söllner et al.,
1993a
) and yeast (Rossi et
al., 1997
) SNAREs also bind three SNAP and Sec17p, respectively,
indicating a conserved behaviour in SNARE:SNAP:NSF complex formation. Even
though NSF ATPase activity is essential for breaking apart the SNARE complex,
the comatose equivalent of CHO NSF (274G
E),
deficient in ATPase activity, could nonetheless mediate membrane fusion
(Müller et al., 1999
).
These data imply that NSF possibly functions in steps other than the
well-studied ATPase-dependent SNARE disassembly-mediated reactions
(Schwarz, 1999
).
The precise function of NSF in membrane fusion is controversial.
NSF-mediated SNARE disassembly might precede fusion
(Banerjee et al., 1996) or in a
priming step after docking (Kawasaki et
al., 1998
), or after fusion to initiate another cycle of fusion
(Littleton et al., 1998
,
2001
;
Schweizer et al., 1998
).
Mutations in Drosophila NSF1 result in the accumulation of docked
vesicles (Kawasaki et al.,
1998
). This may result from undissociated SNARE complex on
vesicles that are recycled, or on the plasma membrane formed after fusion in
comatose flies. Such tangled SNAREs would be incapable of forming
productive v-t-SNAREs in trans. NSF-mediated disassembly in wild-type
synapses would relieve this constraint after fusion, such that v-SNARE from
SNARE bundles formed after fusion are recycled efficiently on endocytosed
vesicles, leaving t-SNAREs on plasma membrane free to pair with incoming
v-SNARE. Since these events can be viewed as `beginning' and `end' reactions
within one round of fusion, prevailing data support the action of NSF not
during fusion but in disentangling SNARE bundles residing on the same membrane
(cis-SNAREs), formed after fusion (Littleton et al.,
1998
,
2001
;
Tolar and Pallanck, 1998
).
This interpretation is also consistent with Sec18p in yeast vacuolar fusion,
where it mediates an ATP-dependent priming of vacuoles even before fusion
partners come in contact with each other
(Ungermann et al., 1998
;
Wickner and Haas, 2000
).
Structural studies on NSF (Lenzen et
al., 1998; May et al.,
1999
; Yu et al.,
1998
,
1999
), Sec18p
(Babor and Fass, 1999
) and p97
(Rouiller et al., 2000
;
Zhang et al., 2000
) are
providing new avenues for improving our understanding of how conformational
changes associated with nucleotide binding and hydrolysis might possibly
disassemble SNAREs (see Dalal and Hanson,
2001
; Hanson et al.,
1997
; May et al.,
2001
). Similar to neuronal activity-dependent phosphorylation of
synaptic proteins (Greengard et al.,
1993
), vertebrate NSF could be phosphorylated by protein kinase C
(PKC) in synaptosomes (Matveeva et al.,
2001
), which is consistent with our previous proposal
(Pullikuth and Gill, 1999
). It
remains to be seen how such modifications can modulate the several membrane
fusion reactions that are catalyzed by NSF.
NSF is critical for synaptic transmission, since vesicles accumulate
(Kawasaki et al., 1998) and
lead to paralysis when its function is impaired
(Pallanck et al., 1995a
;
Siddiqi and Benzer, 1976
).
Apart from its better-understood role in neurotransmitter release, NSF
mediates trafficking or proper insertion of glutamate receptor subunits at
postsynaptic sites (Osten et al.,
1998
; Song et al.,
1998
). Further, NSF interacts with ß2-adrenergic
receptor (Cong et al., 2001
)
and ß-arrestin 1 (McDonald et al.,
1999
), probably to facilitate receptor internalization and
recycling. This indicates that NSF might have varied functions depending on
the local cellular context. Despite its established role in neurotransmission,
little is known about NSF distribution and function in insect endocrine cells.
Our histochemical analysis of MsNSF expression suggests that, in M.
sexta, NSF expression is under developmental control. The specific
enrichment of MsNSF in Type IIa4 cells suggests that NSF plays a
physiologically important role in later development. We did not detect
significant colocalization of neuropeptides and hormones with MsNSF
enrichment. Thus, MsNSF expression in Type IIa4 cells is a novel
phenotype. It has been speculated that TypeIIa4 cells, which
produce diuretic hormone (DH) in larvae, are atrophied during pupal
development since DH production shifts to a cluster of smaller cells derived
from a common neuroblast (Veenstra and
Hagedorn, 1991
). Incidentally, DH immunoreactivity was the only
means of readily identifying these cells. Our observation that NSF-IR
localizes to Type IIa4 cells in late pupal and adult stages
suggests that these cells are not atrophied but in fact might govern crucial
behavior in development, probably through an NSF-dependent mechanism that
regulates secretion from CC.
Another important aspect of NSF expression is at the level of sites known
to release hormones and neuropeptides that are synthesized in the brain. If
NSF were a constitutive member of the release machinery then one would expect
its expression to be more or less uniform in all identified sites of hormone
release. The fact that eNSF-IR is found only in CC and not in CA strongly
indicates that its preferential role is mediating the release of a subset of
hormones that are synthesized in the brain and released from CC. Hormones
secreted from CA might thus use machinery distinct from NSF that is
characterized here, or might be NSF-independent. Of the sequenced genomes,
Drosophila is the only organism so far to have two distinct NSF
homologues, dNSF1 and 2. dNSF1 is required for early adult development whereas
dNSF2 is required for early larval development. The expression patterns of
neither of them have been adequately examined in endocrine cells, even though
one can genetically rescue the defect in the other
(Golby et al., 2001). The D1
domain, towards which our antibody was raised, is highly conserved (>80%)
in both dNSF homologues and would be expected to react with such homologues if
present in M. sexta. MsNSF expression in a subset of enteric
endocrine cells indicates that MsNSF might play a role in the regulation of
processes emanating from factors produced in the midgut. The strategically
located pediculate cells reactive for MsNSF are exposed to the hemolymph and
thus could receive information from hemolymph-borne factors governing several
aspects of development and homeostasis.
In summary, we have provided in vivo evidence for the functional
role of MsNSF. Our results experimentally verify that MsNSF could functionally
replace Sec18p in vivo. This supports the findings of Steel et al.
(1999), who showed that
purified Sec18p could participate in Ca2+-triggered exocytosis in
chromaffin cells. The developmental regulation of NSF expression in
neurosecretory cells points to a novel role of NSF in peptidergic pathways
dictating developmental aspects of M. sexta. Enrichment of MsNSF in
enteric endocrine cells suggests that NSF might play a preferential role in
hormonal secretion in a subset of cells, whereas the remainder might use
either NSF-independent pathways or other NSF-like molecules distinct from the
one characterized here. We hope that this study will encourage the use of
yeast as a tractable genetic system to understand key roles for proteins in
secretion from organisms where cell-free assays are not currently available or
are difficult to develop.
![]() |
Acknowledgments |
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