1 University of Massachusetts, Department of Biology, Morrill Science Center,
Amherst, MA 01003, USA
2 Division of Neurosciences, Beckman Research Institute of the City of Hope,
1450 E.Duarte Road, Duarte, CA 91010, USA
* Author for correspondence (e-mail: rmurphey{at}bio.umass.edu)
Accepted 1 May 2003
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SUMMARY |
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Key words: Synaptogenesis, Semaphorin, Endocytosis, Dynamin, Drosophila
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INTRODUCTION |
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The complexities of the signaling pathways that regulate synapse formation
are only now being recognized. At the Drosophila neuromuscular
junction, the trafficking of TGFß and Wingless/Frazzled have recently
been linked to synaptic differentiation. In the case of the TGFß the role
of membrane trafficking was emphasized because proteins in the endosomal
system affect signaling and their loss led to a large over growth of the
presynaptic terminal (Sweeney and Davis,
2002). Similarly Wingless/Frazzled and its trafficking has been
linked to maturation of the presynaptic terminal
(Packard et al., 2002
). In
both cases the first step in the process is the dynamin-dependent endocytosis
of the receptor. Thus, we reasoned that it might be useful to disrupt
endocytosis at various times during the transition from growth cone to synapse
and determine whether there are critical periods during which endocytosis is
crucial to synapse formation, maturation or stabilization.
In Drosophila, there is suggestive evidence that blocking
endocytosis during particular stages of development of the giant fiber (GF)
system disrupts synaptogenesis (Hummon and
Costello, 1987). The original mutation in dynamin was a
temperature-sensitive paralytic mutant in Drosophila called
shibire, which was linked to synaptic transmission
(Koenig and Ikeda, 1996
;
van der Bliek and Meyerowitz,
1991
) and to the regulation of signaling systems
(Di Fiore and De Camilli,
2001
). The mutant functions as a dominant negative at the
restrictive temperature and can be used to block endocytosis on a wild-type
background (Damke et al.,
1995a
; Damke et al.,
1995b
; Kim and Wu,
1987
). The problem with the original experiments that examined
development of the giant fiber system was that temperature shifts blocked
endocytosis in all cells and made the interpretation of the results difficult
(Hummon and Costello, 1987
).
The temperature-sensitive allele of shibire has now been cloned into
a P-element (UAS-shits) and can be targeted to cells of
interest in Drosophila, thereby gaining both temporal and cell
specific control over endocytosis
(Kitamoto, 2002
). In addition,
a panel of Gal4 gene targeting constructs for the GF system is
available, which allow targeted expression to different components of the GF
system (Allen et al., 1998
;
Allen et al., 1999
;
Allen et al., 2000
;
Godenschwege et al., 2002a
).
This provides a method to assess the required timing for endocytosis in the
developing nervous system.
In the present report, we targeted expression of the temperature-sensitive
dynamin to the GF system and blocked endocytosis during development. Blockade
of endocytosis at different times revealed four phases of sensitivity and
temperature shifts during each phase have a different consequence for the
development of the GF system. We previously demonstrated that Semaphorin 1a
(Sema1a) was involved in assembly of the GF system
(Godenschwege et al., 2002a)
and we now show that defects were induced primarily during the period of
synapse formation. Finally, when UAS-sema1a was co-expressed with
UAS-shits and endocytosis was blocked at different times,
the two effects interacted, suggesting that one of the proteins being
regulated by endocytosis was Sema1a.
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MATERIALS AND METHODS |
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Three P[Gal4] drivers were used to drive various constructs in the GF
system. The enhancer known as P[Gal4]-A307 an insert on the second chromosome
(Allen et al., 1998) was
expressed primarily in the GF and weakly in other components of the system
including the tergotrochanteral motoneuron (TTMn) and the dorsal longitudinal
motoneuron (DLMn) and the muscles of the thorax particularly the
tergotrochanteral muscle (TTM) and the dorsal longitudinal muscle (DLM). The
enhancer known as P[Gal4]-c17 an insert on the second chromosome
(Allen et al., 1999
;
Godenschwege et al., 2002a
) is
expressed in the GF but not in the postsynaptic neurons and it can be used for
exclusively presynaptic expression. Finally, the postsynaptic cells were
labeled by the P[shakB-Gal4] construct, a fragment of the gap junction
promoter fused to Gal4 and inserted on the 2nd chromosome
(Jacobs et al., 2000
). This
construct is expressed in TTMn, DLMn, a variety of other motoneurons and the
PSI (peripherally synapsing interneuron). The UAS-shits on
the third chromosome was obtained from Kitamoto and expressed under control of
one of the three Gal4 constructs
(Kitamoto, 2001
;
Kitamoto, 2002
). A UAS-sema1a
construct on the 2nd chromosome was used to express Sema1a
(Yu et al., 1998
). Finally, a
UAS-lacZ construct on either chromosome 1, 2 or 3 was expressed under control
of the relevant enhancer and expression revealed immunohistochemically.
Blocking endocytosis
Endocytosis was compromised by shifting specimens to the restrictive
temperature (30°C in most experiments). Although development speeds up
with temperature it shows a biphasic curve and slows at 30°C to a rate
similar to that at 22°C (Ashburner,
1989) so this temperature shift has relatively small effect on the
rate of development. To show that the construct was blocking endocytosis, we
expressed UAS-shits in the motoneurons under the control
of A307 and then temperature shifted the specimens acutely. We mounted the
specimens on wax on a pelltier battery which allowed a rapid shift of
temperature (less than 3 minutes) from 22°C to 30°C. Recordings were
obtained before during and after the temperature shift. This blocked synaptic
transmission within minutes at the DLM, in a manner parallel to that seen in
early experiments on the original shibire mutant
(Koenig and Ikeda, 1996
), and
at the TTM.
Physiology
The physiological methods are standard
(Oh et al., 1994;
Tanouye and Wyman, 1980
) and
we have modified them only slightly (Allen
et al., 1999
; Godenschwege et
al., 2002b
). The specimens were anesthetized by placing them on
ice and then mounted on soft wax. Tungsten electrodes were used to stimulate
the GF, and glass electrodes to record from the muscles. The data was recorded
using P-Clamp software (Axon Instruments).
In specimens (A307/+;UAS-shits/+) reared at 22°C and never given any form of temperature shift, we were unable to obtain recordings from the TTM. Sections of the thorax demonstrated that the TTM muscle was small or absent in these specimens although the DLM was normal. The A307 construct is known to be expressed in the muscles of the thorax and we assume that the expression of the P[UAS-shits] in the muscle at 22°C, throughout development disrupts normal muscle development, suggesting that shits has an effect at the non-restrictive temperature. When A307; UAS-shits was reared at 18°C the TTM muscles of control specimens were intact and we used this rearing temperature for the physiological experiments described below. We examined the anatomy of the GFs at both rearing temperatures and in spite of the absence of TTM muscles at 22°C and their presence at 18°C we saw no difference in the anatomical phenotypes of the GFs after temperature shifts.
Immunohistochemistry
The CNS was dissected from adults or pupae at the appropriate stage and
standard methods were used to reveal lacZ in the various neurons
(Allen et al., 1999;
Godenschwege et al.,
2002b
).
Image processing
Images of selected whole mounted specimens were captured using a SPOT
digital camera (Diagnostic Instruments, Sterling Heights MI) and imported into
Adobe Photoshop 5.0 software. Montages were constructed using the clone tool
to show a two-dimensional image of the three dimensional neurons.
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RESULTS |
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The dye injection results provided a very detailed picture of the neurons but rather little about the temporal variability of these developmental processes. By combining the c17 enhancer and the shakB-Gal4 element, we labeled the pre and postsynaptic cells simultaneously and this provided an indication of the variability in GF growth. We focused on the period when the GF was reaching the target area and collected a number of specimens at this time. The results showed that at 33% of pupal development the TTMn dendrite had always reached the midline but there was considerable variability in the position of the GF axon terminal; approximately half (16/33) of the GFs had contacted the TTMn and half had not. In a typical specimen at this stage, both GFs can be seen adjacent to the midline glia but only one has reached the dendrite of the TTMn (Fig. 1B1).
Blocking endocytosis at different times produces three distinct
axonal phenotypes
In order to begin to assess the role of endocytosis in the GF and provide
an outline of critical periods in pupal development we blocked endocytosis
during various phases and assessed GF structure and function. The
P[UAS-shits]
(Kitamoto, 2001;
Kitamoto, 2002
) was expressed
under control of the A307 (Fig.
1). We used the A307 because it was expressed strongly in the GF
throughout the pupal stages and this allowed us to compromise endocytosis at
anytime during pupal development and assess the effects on GF growth.
Endocytosis was compromised by shifting specimens from the permissive
temperature (18°C or 22°C) to the restrictive temperature (30°C)
at various times during pupal development. The restrictive temperature
disrupted the pupal development of the GF system and the phenotype in the
adult correlated with the timing of the temperature shift. In parallel
physiological experiments, we examined the efficacy of the synaptic
connections from GFs to the motoneurons by stimulating the GF in the brain and
recording from the TTM and DLM muscles in adults
(Allen et al., 1999
;
Tanouye and Wyman, 1980
;
Thomas and Wyman, 1984
). Here,
we focus on the monosynaptic GF-TTMn response and use the response of the
polysynaptic GF-DLM pathway to determine the percentage of GFs making a
connection, demonstrating their presence in the target area. Four phases of
sensitivity during development of the GF system were identified. For narrative
purposes, we describe these phases as having distinct boundaries although
there are continuous transitions between the stages (Tables
1,
2).
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Phase III (stabilization)
Temperature shifts at 62.5 or 75% of pupal development had minor effects on
the GF anatomical phenotype; typically the bend was present and normal in size
although it may contain irregularities in shape
(Fig. 2A3). However,
physiologically, these specimens were defective in the adult, the TTM
exhibited long latencies and low following frequencies
(Table 1). In brief, during
this phase the GF anatomy had stabilized and was resistant to the blockade of
endocytosis, while the synapse physiologically was still sensitive to
temperature shifts.
Phase IV (mature synapse)
Finally, very late temperature shifts (e.g. at 87.5% of pupal development)
had no impact on the GFs or their ability to drive the motoneurons.
The anatomy of the GFs was indistinguishable from controls (Fig. 2A4) and functionally there were no significant defects on latency or following frequency for either TTMn or DLMn (Table 1).
Dynamics of GF retraction and regeneration Axon retraction
In order to appreciate the dynamics of the anatomical changes, we used
P[Gal4]-A307 to express UAS-shits and examined the axonal
phenotypes before the temperature shift, immediately after the temperature
shifts as well as at later times (Fig.
3, Table 2). The GF
grew into the thorax at the beginning of pupal development
(Allen et al., 1998;
Phelan et al., 1996
) and when
dissected immediately after the temperature shift, the GF anatomy could be
reliably observed. Most of the GF axons terminated in `retraction bulb'
(Fig. 3A2, arrows) often
exhibiting a long slender extension towards the target
(Bernstein and Lichtman, 1999
).
These retraction bulbs were located in the connective (36% of the GFs) or the
first thoracic neuromere (53% of the GFs) and a minority terminated in the
brain (Table 2). By contrast,
GFs treated in the same way but examined as adults exhibit overgrowth
(Fig. 3A3) and the terminals
were located in the second thoracic neuromere (53% of the GFs) and often
extended to the third thoracic neuromere (46% of the GFs), demonstrating
regeneration of the axons after return to the permissive temperature. When the
axon was examined at various stages after the temperature shift axon growth
recovered and the overgrowth phenotype was detected as soon as 48 hours after
the temperature shift ended (at 75% of pupal development). This suggested that
the regenerating axons exhibited the overgrowth defect as soon as they reached
the target region.
|
At 50% of pupal development the GF is known to be dye-coupled to a number
of its targets, including the TTMn (Jacobs
et al., 2000; Phelan et al.,
1996
), and the presynaptic terminal is beginning to take on the
adult appearance. Temperature shifts at this stage exhibited less dramatic
retraction and immediately after the temperature shift the presynaptic
terminal had retracted only slightly from the target area
(Fig. 3C2). The defect
developed progressively and more GFs were anatomically defective in the adults
than immediately after the temperature shift
(Table 2). When examined in the
adult stage the presynaptic terminal had expanded but there were often defects
in its structure and the bend was not as large as in control specimens
(Fig. 3C3). The synapse
retraction was more complete if the temperature shift was longer in duration
(48 hours rather than 24 hours, data not shown). These results demonstrated
that this immature synapse could be retracted even after dye-coupling had
occurred at 40% (Jacobs et al.,
2000
) but could usually not be regenerated after the temperature
shift.
Synapse stabilization
Finally, temperature shifts during the final third of pupal development
caused minor anatomical defects in the synaptic terminal but no retraction.
For example, when exposed to the restrictive temperature beginning at 67% of
pupal development and dissected immediately after the temperature shift, the
axon extended laterally as it would in wild-type specimens. The terminal was
often irregular in shape and contained swellings at various places along the
terminal but the laterally directed bend was usually present
(Fig. 3D2). When dissected in
the adult stage the result was very similar. Most terminals exhibited the
lateral bend and approximately half of these synaptic terminals were distorted
by swellings in the bend or at a site just anterior to the bend. When the
temperature shift was extended to 48 hours, there was no additional synapse
retraction indicating that the synapse had stabilized after 67% and could not
be retracted from the target area after this stage.
Controls for the temperature shifts
A variety of controls were carried out to distinguish the effect of
blocking endocytosis from non-specific effects of the temperature shift. We
temperature shifted specimens carrying the A307 enhancer and the
UAS-lacZ but no UAS-shits at various times but
all GFs appeared wild type. Thus, the temperature shift per se did not cause
detectable anatomical defects in GF. To assess the possibility that the
UAS-shits construct was `leaky', we applied temperature
shifts to specimens carrying the UAS-shits construct but
no Gal4 driver. All specimens treated in this way exhibited wild-type
physiology demonstrating that the UAS-shits construct must
be driven by Gal4 in order to have its effects. Finally, we reared specimens
carrying the UAS-shits construct and a Gal4 driver without
temperature shifts and this led to wild-type anatomy in 90% of the cases
(Table 3).
|
Temporal aspects of Sema1a function
It is well known that the P[Gal4] enhancers and the UAS-drivers are
temperature sensitive and we took advantage of this property to determine the
critical periods for Sema1a. In specimens (c17/UAS-sema1a) reared at
18°C and never temperature shifted most of the axons were normal in
structure (83% wild type) and function (67% wild type) similar to that seen
for specimens reared at 22°C
(Godenschwege et al., 2002a).
When specimens were temperature shifted to 30°C during synapse formation
(37.5% of pupal development), the axon terminal typically lacked the bend in
adult flies and physiologically all GF-to-TTMn contacts exhibited long
latencies and/or low following frequencies
(Fig. 4A1,
Table 3). When the temperature
shift occurred at 50% of pupal development, a minority were anatomically wild
type and a few were physiologically normal
(Table 3,
Fig. 4A2). By contrast,
temperature shifts before or after these times had only minor effects in
comparison to non-temperature-shifted control specimens
(Table 3). These results
confirmed that Sema1a was disruptive for synapse formation and suggested that
it must be removed in order for synapse formation to proceed normally. In
addition, these new results demonstrated a critical period for removal of
Sema1a during the period of synapse formation and showed that its acute
presence during phase II had a permanent effect that prevented regeneration of
a functional synapse.
|
Co-expression of UAS-sema and
UAS-shits
In order to determine whether Sema1a trafficking/signaling was dependent on
endocytosis, we used P[Gal4]-c17 to co-express UAS-shits
and UAS-sema1a and searched for interactions. It was clear that even
without a shift to the restrictive temperature, there was an interaction,
because only half of the specimens exhibited anatomically normal GFs and the
remainder were bendless-like or did not reach the target area
(Table 3). This suggested that
both constructs were expressed at low levels at 18°C and indicated that
UAS-shits was having an effect even at the permissive
temperature of 18°C, an effect that was detected in double mutant
specimens but not when UAS-shits was expressed alone. The
physiology was correlated with this as approximately half were wild type and
half were mutant (Table 3).
Temperature shifts during the period of synapse formation (phase II) caused dramatic defects in the presynaptic terminal (Table 3). When UAS-sema1a and UAS-shits were co-expressed and the temperature shifted at 37.5% of pupal development, nearly all GFs were defective; approximately half (44%) of the GFs terminated in the thorax and were anatomically defective (Fig. 4C1) and most of the remaining axons terminated in the brain (Table 3). When the temperature shift began at 50% of pupal development, most axons exited the brain but the presynaptic terminals in the thorax were defective (Fig. 4C2, Table 3). The disrupted axon terminals were unusually large, and often filled with membrane-bound vesicles that excluded lacZ (Fig. 4C1 and Fig. 4C2). Small vesicles were occasionally observed when either construct was expressed alone (data not shown) but the large vesicles in the co-expression experiment highlight the possibility that membrane trafficking has been disrupted when both constructs were expressed. The physiological results were consistent with these anatomical findings. Although the axon terminals were connected to one or the other motoneurons, all connections were physiologically defective (Table 3). Finally, the results also show that the defect is a progressive degenerative effect as these large vesicular structures only emerge after a delay. The large vesicles were never seen immediately after the temperature shift and were only seen in adults after temperature shifts at 33% or 50% of pupal development (Table 3).
Finally, the interaction of shibirets and sema1a was further supported by temperature shifts that began at 62.5% of pupal development (phase III). When sema1a and shits were co-expressed by the weak c17 driver and temperature shifted at this pupal developmental stage, none of the specimens exhibited a bend when dissected directly after the temperature shift, suggesting all immature synapses had retracted (Table 3). A few GFs were able to regenerate anatomically after the temperature shift but physiologically, not a single fully functional giant synapse was restored (Table 3). A temperature shift in phase III was not able to induce the retraction of the giant synapse when UAS-shits was driven with the strong driver A307 and only minor effects on the GF anatomy were seen when either construct was expressed alone with c17 and temperature shifted. These findings demonstrate that sema1a and shibrets have a synergistically enhanced ability to induce the retraction of a synapse. In summary, comparison of the co-expression experiments with those for either UAS-shits alone or UAS-sema1a alone supports the idea that the two are interacting and suggests a role for membrane trafficking in Sema1a signaling.
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DISCUSSION |
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Four phases of circuit assembly revealed by heterochronic growth
In the present experiments, targeted blockade of endocytosis had direct
effects on axon growth and retraction, presumably by disrupting the recycling
of membrane in a rapidly growing axon terminal
(Diefenbach et al., 1999).
Previously, neurons from mutant shits1 animals were grown
in culture and a shift to the restrictive temperature caused collapse of
growth cones, cessation of axon outgrowth and axon retraction. Shifting back
to the permissive temperature led to a resumption of growth and a rebound of
growth rate (Kim and Wu,
1987
). The temperatures used and the temperature shift paradigms
employed in vitro were identical to those we used to assay the timing of
developmental events of the giant fiber in vivo. When we challenged the GF at
various times during pupal development by blocking endocytosis, we identified
four phases in the GF development: an early pathfinding phase, an intermediate
phase of synaptogenesis, a late stabilization process and, finally, a mature
synapse.
When we blocked endocytosis during pathfinding; the axons retracted during the temperature shift and when returned to the permissive temperature regenerated and overgrew the target area (phase I, Fig. 1A). By contrast, temperature shifts which correspond to the time that the GF is being transformed from growth cone to synapse (phase II) produced a different effect. GFs retracted but when examined as adults the axons did not overgrow the target area but rather stopped in the target area and lacked the lateral bends. The initial effects induced by blocking endocytosis during both, phase I and II, were likely to be caused by the retraction of the axon and the subsequent heterochronic growth of the GF. However, the difference of the responses (overgrowth versus bendless-like) between phase I and II cannot be attributed directly to the block of endocytosis, but are more likely to be attributed to the different developmental states of the GF when heterochronic regeneration occurred. One relevant difference may be that in phase I most GFs have not contacted the target area and in phase II most GFs have contacted the targets. This means that the heterochronic growth induced in phase I results in naïve GFs that approach the target area with a delay, while heterochronic growth in phase II results in the re-generation of `experienced' GFs. Possibly the GF loses its ability to regenerate the GF-TTMn synapse after it has contacted the target resulting in the bendless-like phenotype.
Finally, blockade of endocytosis in phase III revealed a distinct defect.
The function of the synapse was disrupted by temperature shifts although the
structure remained normal. This distinguished a stabilized synapse from a
mature synapse. Possibly the block of endocytosis during phase III disrupts
trafficking of receptors/ligands that are involved in maturation of the giant
synapse. For example, Fasiclin 2 and Wingless/Frazzled have been shown to be
required for maturation of the neuromuscular junction and correct
dynamin-dependent trafficking is required for normal synaptogenesis
(Davis et al., 1996;
Packard et al., 2002
;
Schuster et al., 1996
).
Critical periods for Sema1a function
We have previously suggested that Sema1a must be removed from the
presynaptic terminal in order for synaptogenesis to proceed correctly, but the
exact timing and mechanism for these events were not examined
(Godenschwege et al., 2002a).
In the present report, we show that the acute presence of Sema1a during
synapse formation had a lasting effect that prevented the regeneration of a
functional synapse. We examined the temporal aspects of Sema1a function,
independent of endocytosis, by taking advantage of the temperature sensitivity
of the UAS constructs. Overexpression of Sema1a during synapse formation
(phase II) caused the majority of axons to terminate in bendless-like
structure and exhibit weak synaptic connections
(Table 3), while the acute
presence of Sema1a in phase I or phase III had only minor effects. This
suggests that removal of Sema1a is crucial for synaptogenesis. The sensitivity
to Sema1a overexpression overlapped the time that the GF first contacted its
targets and becomes dye-coupled to them
(Phelan et al., 1996
)
suggesting that Sema1a plays a role in the transition from growth cone to
synapse. Interestingly, the bendless mutant causes phenotypes similar
to those seen when Sema1a is overexpressed in the present experiments
(Thomas and Wyman, 1984
). When
bendless was cloned and shown to be a ubiquitin conjugase the authors
speculated that the bendless mutant may be affecting the lifetime of
Sema1a on the GF growth cone (Muralidhar
and Thomas, 1993
; Oh et al.,
1994
). The finding that Sema1a trafficking is involved in the
assembly of the GF-TTMn synapse and the recent realization that ubiquitin can
function to regulate trafficking of membrane proteins
(Murphey and Godenschwege,
2002
) suggest that Sema1a trafficking may be regulated by
Bendless.
Endocytosis and Sema1a signaling
Endocytosis plays an important role in ligand-dependent receptor responses
that serve as a mechanism for the regulation of signal strength in a variety
of signaling pathways (Di Fiore and De
Camilli, 2001; Hicke,
1999
). We propose that during the transition from growth cone to
synapse, Sema1a, which functions as a receptor on the GF growth cone,
encounters its ligand and this slows the progress of the growth cone as a
first step in the transition (Godenschwege
et al., 2002a
). However, the repulsive signaling of Sema1a must be
downregulated because it is disruptive for subsequent events in the formation
of the synapse and it is therefore normally removed through a
dynamin-dependent receptor-mediated endocytosis. When UAS-sema1a was
combined with UAS-shibirets in a genetic interaction
experiment, simultaneous overexpression of Sema1a and the block of endocytosis
exaggerated the disruptive effects of Sema1a. One effect was greater
retraction of the axon presumably by enhancing the total amount of the
repulsive receptor (Sema1a) present on the surface of the presynaptic cell
(Table 3). A second effect was
the accumulation of large vesicles in the axon terminal. Our interpretation is
that the unusually high levels of Sema1a at the surface activated excessive
receptor-mediated endocytosis. This may cause a vesicular `traffic jam' in the
growth cone, thereby disrupting the ability to carry out normal functions.
These vesicular traffic jams are consistent with other experiments on the GF
system that show similar phenotypes. For example, blocking retrograde
transport by expression of a truncated version of the P150Glued
component of the dyneindynactin motor also caused the formation of large
vesicles in the GF terminal (Allen et al.,
1999
). Although we cannot directly link the vesicles seen in these
various genotypes to each other, the common phenotype makes it seem likely
that we are interrupting a common membrane trafficking pathway involved in
synapse formation. Markers for various aspects of the endosomal system in
Drosophila (Sweeney and Davis,
2002
) will eventually allow us to identify the origin of these
vesicles and link the various genotypes together in a model of receptor
trafficking and synapse formation.
In vertebrate neurons, semaphorin signaling has been linked to endocytosis
during growth cone guidance and growth cone collapse
(Fournier et al., 2000;
Jurney et al., 2002
). Sema3a
serves as a ligand for the plexin/neuropilin receptor complex and has been
shown to stimulate endocytosis during growth cone collapse. Moreover this is a
Rac1-mediated process as Sema3a and Rac1 are associated with vesicles after
Sema3a treatment (Fournier et al.,
2000
) and Rac1 is required for endocytosis of growth cone membrane
during growth cone collapse (Jurney et
al., 2002
). Although Sema3a is working as a ligand in vertebrate
neurons and Sema1a is working as a receptor in the GF, there are a number of
striking parallels between the vertebrate work and the Drosophila
work. In both cases, semaphorin and endocytosis are linked and in both cases
Rac1 is involved in growth cone structure and behavior. We demonstrated
elsewhere that overexpression of the small GTPase Rac1 disrupted the
termination of the GF and caused the accumulation of large vesicles in the
terminal (Allen et al., 2000
).
Although we did not experimentally link Rac1 to the semaphorin effects, the
similarity between the GF phenotypes in these various experiments is
consistent with the vertebrate work. The involvement of semaphorins, Rac1 and
endocytosis in growth cone repulsion in vertebrate neurons and in the
transition to synapse formation in the Drosophila GF system
highlights the similarities between the systems. As synapse formation requires
that growth cones slow or stop as they invade a target region, it seems likely
that the growth cone guidance machinery has been commandeered to regulate the
initial stages of synaptogenesis.
Surprisingly, the appearance of large vesicles in the GF in the interaction experiment between Sema1a and endocytosis was delayed with respect to the temperature shift as no vesicles were detected immediately after the temperature shift but rather the vesicles emerged as pupal development proceeded (Table 3). There are numerous suggestions that defects in membrane trafficking are linked to neurodegeneration and that these vesicles in the GF may be a prelude to synaptic degeneration; we are exploring this possibility.
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ACKNOWLEDGMENTS |
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REFERENCES |
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