Biology and Chemistry Research
Drosophila p21-activated kinase 3 in Glia Interacts with flower in Neurons to Regulate Synapse Structure and Function Daniel Ren
ABSTRACT
The primarily neuronally expressed gene flower plays an important role in synapse development, mediating the targeted release of Ca2+ at presynaptic terminals in order to couple endocytosis and exocytosis, allowing for proper synaptic vesicle fusion and neurotransmitter release. To gain insight into how flower is regulated, we examined the predominantly glial gene p21-activated kinase 3 (pak3). pak3 is known to interact with spastin, an AAA ATPase involved in microtubule severing, which is important to synapse development. Since both flower and spastin affect synapse formation, we hypothesized that pak3 also interacts with flower. To test this idea, we dissected Drosophila larvae with pak3 and flower mutations and inspected the synapses at their muscle 4 neuromuscular junctions. Here, we show that whereas pak3 mutants show only minimal synaptic defects and flower mutants have major deficiencies, when both genes are simultaneously mutated, loss of pak3 significantly enhances flower mutant synaptic phenotypes. Thus, pak3 and flower interact synergistically to regulate synapse structure and function. Additionally, this supports the theory that glia are essential nervous system components and play key roles in proper nervous system functions. [5][6][7][8]. Each neuron’s axon contains a number of Introduction presynaptic terminals, or boutons, where neurotransmitter is released from the neuron into the muscle cell. The development and maintenance of neuronal Neurotransmitter receptors on the muscle cells then synapses is a complex process that involves regulation receive the signal, which causes positive ions to enter and from a variety of proteins which are encoded by specific depolarize the muscle cells, leading to various physiological genes. Much is currently known about the structure and effects in the cells [9][10]. However, regulation and function of synapses, however, less is known about the maintenance of these synapses is much more complex, and molecular pathways and mechanisms that shape synapse involves not only neurons but glia as well [11][12][13]. development and allow synapses to function properly Previous studies involving synapse regulation have [1]. What is particularly important and intriguing is how looked towards p-21 activated kinase 3, or pak3, a loosely synapses at neuromuscular junctions (NMJs) are regulated evolutionarily conserved Drosophila gene expressed in glia, and maintained, since these connections ultimately lead to which is thought to regulate the actin cytoskeleton in glial proper motor function and coordination of the organism cells [14][15]. When pak3 alone is mutated, there are only [1][2]. mild effects on synapse structure and function. However, In this report, we use the model organism Drosophila pak3 is also known to interact with spastin, a functionally melanogaster to study the NMJs. Drosophila provides conserved gene which encodes an AAA ATPase that is a plethora of benefits for the researcher, particularly involved in microtubule-severing in neurons. Spastin when studying synaptic transmission. Benefits of the serves to sever microtubules to allow for proper axonal Drosophila NMJ as a model synapse include the easy development and synapse formation, particularly affecting manipulation of genetics and observability of phenotypes, the structure and function of boutons at NMJs [16][17] its accessibility to the powerful experimental technique of [18]. Mutations in spastin lead to an increased number immunocytochemistry, its close resemblance to vertebrate of boutons at the NMJs, a decreased average bouton size, glutamatergic synapses, and finally, its incredible plasticity a more “bunched” structure, and a reduced amplitude of and dynamic nature [1][3][4]. Additionally, Drosophila has evoked responses, or reduced functionality (Sherwood et a fairly simplistic body layout, which allows us to easily al., 2004). However, when pak3 is mutated in addition to examine its synapse structures at multiple NMJs per animal spastin, the loss of functional pak3 completely suppresses [1][4]. In this particular investigation, we look at muscle 4 spastin mutant phenotypes. This is interesting because NMJs because they are easily quantifiable in comparison to it shows that pak3 could potentially play a major role in the more complex neuronal arbors at other muscles. Most synapse regulation [19]. importantly though, Drosophila is ideal for a model system Another evolutionarily conserved gene, flower because it shares many evolutionarily conserved genes and (fwe), encodes an important Ca2+ channel which regulates biochemical nervous system pathways with vertebrates – endocytosis and exocytosis in neurons and allows for including humans – which makes it an effective tool to sustained neurotransmission in presynaptic terminals [20] study neurobiological processes [4]. [21]. To do this, Flower proteins localize at the periactive Currently, we know that various neuronal proteins, zones during synaptic fusion, which allows for the antibodies, and enzymes interact at the active zones of neurotransmitter signal to be properly transferred synapses to promote vesicular neurotransmitter release Volume 3 | 2013-2014 | 55
Biology and Chemistry Research from the presynaptic cleft of neurons to the receptors on muscle cells. Similar to spastin, flower also affects synapse structure and function at the NMJs, causing numerous extra boutons (often of a small and clustered nature) to be present at the NMJs, which leads to reduced synaptic vesicle function [21]. In this investigation, we desired to find out whether or not pak3 interacts with genes, other than spastin, which affect synapse structure and bouton formation, such as flower. If there is interaction (whether it be positive or negative interaction), it would lend greater support to the theory that pak3 is involved in regulating synapse structure overall (and is not just spastin-specific), as well as impact our current understanding of the role that glia play in structuring synapses and maintaining their proper function and utility.
Materials and Methods Drosophila stocks and genetic combinations To conduct this experiment, various fly stocks with different genotypes were bred to obtain larvae with desired genotypes. In total, there were 3 groups of fly larvae with different loss-of-function mutations. pak3d02472/Df(3R) pak3 heterozygotes had a d02472 PBac insertion in the pak3 gene, which dramatically reduces mRNA expression, and a Df(3R)pak3 complete deletion of pak3, together disabling its actin-regulatory function. fweDB25/fweDB56 heterozygotes had mutations in flower so as to reduce its Ca2+ channeling function. fweDB25, pak3d02472 / fweDB56, Df(3R)pak3 heterozygotes had simultaneous mutations in both genes, disabling the function of both flower and pak3. These fly larvae were obtained by setting up crosses between “parent” flies to obtain the desired genotypes. pak3d02472/Df(3R)pak3 heterozygotes were obtained by crossing pak3d02472/TM6b adult flies with Df(3R)pak3/TM6b adult flies and selecting for pak3d02472/ Df(3R)pak3 larval offspring. Larvae with this genotype were identified based on marker characteristics associated with the balancer chromosome TM6b. The Tubby (Tb) gene has been recombined onto the TM6b chromosome, and is a dominant marker which makes larvae with Tb significantly fatter and shorter than normal. pak3d02472/ Df(3R)pak3 heterozygotes were wild type in length (since they did not have the TM6b balancer chromosome), whereas pak3d02472/TM6b and Df(3R)pak3/TM6b larvae had the balancer chromosome and displayed the tubby trait. TM6b homozygotes died before embryogenesis was complete. fweDB25/fweDB56 heterozygotes were acquired in a similar manner, by selecting offspring from crosses between fweDB25/TM3 Sb Kr-Gal4 UAS-GFP and fweDB56/ TM3 Sb Kr-Gal4 UAS-GFP, based on the marker trait of fluorescence exhibited by larvae with the TM3 Sb Kr-Gal4 UAS-GFP balancer chromosome. fweDB25/fweDB56 larvae were yellowish-grey and non-fluorescent when placed under blue light, whereas heterozygotes with the balancer 56 | 2013-2014 | Volume 3
chromosome emitted a fluorescent green glow under blue light. Similar to TM6b, TM3 homozygotes died before hatching. The experimental group, fweDB25, pak3d02472/ fweDB56, Df(3R)pak3 heterozygotes were attained by selecting non-tubby larval offspring from crosses between fweDB25, pak3d02472/TM6b and fweDB56, Df(3R) pak3/TM6b parent flies. The fweDB25, pak3d02472/ TM6b and fweDB56, Df(3R)pak3/TM6b fly lines were created by genetically recombining the flower mutations and pak3 mutations onto the same chromosome, to allow for the maintenance of a stock. Originally though, all fly stocks came from genetic manipulations of the flies’ genomes. fweDB25 and fweDB56 mutations were introduced by feeding the flies ethyl methanesulfonate (EMS), a chemical mutagen that causes random point mutations in DNA [21]. Flies were then screened for fweDB25 and fweDB56 mutations. The pak3d02472 mutation was obtained using a PBac transposable element. This was injected into the posterior end of early embryos using P-element insertion, which utilizes the enzyme transposase in order to insert the gene mutations into the flies’ genomes [22]. The Df(3R)pak3 deletion was made by genetically inducing flippase (FLP) in a fly, with two PBac insertions that flanked the pak3 coding region (d02472 and e00329). The flippase caused the FRT (FLP-recombination target) sites within the two insertions to line up, make a loop, and recombine, which removed the intervening pak3 coding sequence [19]. Fly husbandry Fly stocks were kept in vials at 25 °C with ample fly food (nutrient-rich carbohydrate and yeast mixture), with approximately 20 flies in each vial. Vials were plugged with cotton balls to keep flies from escaping. Flies were transferred into new vials every 2 weeks when not being used, and every 4 days when being used, in order to ensure that the flies had a hygienic living environment. When setting up crosses to obtain larvae of desired genotypes, selected male and female adult flies were placed in cages (larger than vials) and capped with nutrient-rich grape juice plates with yeast paste. The grape juice plates were made up of a mixture containing agar, Milli-Q water, sucrose, grape juice, tegosept (antifungal agent) and ethanol (to dissolve the tegosept), and were more beneficial to the flies’ health and survival than traditional fly food. Initially the plates were made by using a premade Genesee powder mixture; however, it was determined that using real frozen grape juice concentrate instead of powder created better quality grape juice plates, which allowed fly larvae to survive longer and grow larger before necrosis occurred and the larvae began to die. This allowed for the dissection of slightly larger larvae, which was especially important since the mutations being studied already decreased the life expectancy of the larvae. The fresh grape juice mixture was made by combining
Biology and Chemistry Research 400mL Milli-Q water, 12.0g Difco agar, 13.2g sucrose, 100mL Welch’s grape juice concentrate, and 1g of tegosept dissolved in 10mL ethanol. The mixture was then heated on a hot plate and stirred until its contents dissolved, let cool, and heated again for 5 more minutes. Once the solution cooled to a reasonable temperature (~70 °C), it was pipetted into petri dishes (7mL per dish) and let cool and solidify. The grape juice plates were then capped and stored at 4 °C until they were used. Larval immunocytochemistry Live third instar larvae were filleted and dissected in 1x phosphate buffered saline (PBS, Invitrogen) at room temperature. After dissection, fillets were fixed in 4% paraformaldehyde (PFA) for 35 minutes. Fillets were washed twice quickly in PBS and then placed into eppendorf tubes. They were washed more thoroughly in PBS with 0.2% Triton X-100 (PBST) and placed on a nutator to continuously mix the solution. Fillets were then blocked using a solution composed of PBST with 5% normal goat serum, 0.01% bovine serum albumin, and 0.02% sodium azide (PBTNA) for 1 hour at room temperature. The blocking solution was removed, and primary antibody (rabbit anti-HRP) diluted 1:300 in PBTNA was applied to the fillets overnight at 4 °C. Primary antibody was removed the next day and secondary antibody (goat anti-rabbit Alexa 488, diluted 1:300 in PBTNA) was applied to each tube. The fillets, now incubated in secondary antibody, were nutated at room temperature for 2 hours in the dark, and afterward washed with PBST. With the immunocytochemistry stage being completed, the larval fillets were mounted onto microscope slides in VectaShield mounting medium and sealed with a cover slip and nail polish (acting as an adhesive). Imaging and Quantification NMJ synapses were analyzed under a Zeiss fluorescent light microscope. The primary and secondary antibodies that were previously applied served to stain neuronal membranes green (488 nm) when excited by blue light, which allowed for the visualization of presynaptic bouton structure when viewed under a fluorescent light microscope. Total and terminal boutons were quantified, in order to provide a complete and accurate sense of the synapse structures. Total bouton number was calculated by counting all 1b and 1s boutons at muscle 4 of each hemisegment, no matter their size or structure. Terminal boutons were those at the distal end of an axonal branch, which had no further boutons connected to them. Total bouton number measures overall synaptic growth and terminal number measures the amount of branching in the arbor. Representative images of synapses from each of the genotypes were taken with a Zeiss LSM 510 confocal laser scanning microscope.
Data Analysis After quantifying the total and terminal boutons present in each muscle 4 hemisegment for all of the larvae dissected, the average, standard deviation, and standard error were calculated for each genotype. The total and terminal number of boutons for the three genotypes were then compared to each other, and Student’s t-test (2-tails, homoscedastic) was conducted between fweDB25/fweDB56 and fweDB25, pak3d02472/ fweDB56, Df(3R)pak3 to test for statistical significance.
Results pak3 interacts synergistically with flower Larvae in the pak3 control group, pak3d02472/ Df(3R)pak3, have greatly reduced pak3 gene expression, presumably reducing Pak3 functionality in regulating actin projections. This causes minimal differences in synaptic structure and function compared to wild type larvae [19]. In fweDB25/fweDB56, the flower control group, the flower gene has been mutated so as to compromise its function, reducing the ability of the Flower-mediated Ca2+ channel to allow synaptic vesicle fusion. This causes a significantly increased amount of total and terminal boutons [21]. fweDB25, pak3d02472/fweDB56, Df(3R)pak3 is the experimental group, in which both pak3 and flower have been mutated, effectively eliminating the function of both. In this case, the total and terminal bouton counts at the NMJs are higher than those of the controls, indicating that some relationship between pak3 and flower exists. Figure 1 displays the average total and terminal bouton numbers for all three larval genotypes.
Figure 1. effects of loss-of-function gene mutations on total and terminal bouton counts: this table displays the average number of total and terminal boutons at the muscle 4 NMJs of fly larvae for all three genotypes tested. The experimental group, fweDB25, pak3d02472/ fweDB56, Df(3R)pak3, in which both pak3 and flower have been mutated, has higher average total and terminal bouton counts compared to both of the controls (pak3d02472/Df(3R)pak3 and fweDB25/fweDB56). For terminal bouton counts, the p-value calculated from Student’s t-test between fweDB25/fweDB56 and fweDB25, pak3d02472/ fweDB56, Df(3R)pak3 is .037, which is below Volume 3 | 2013-2014 | 57
Biology and Chemistry Research .05, the cutoff for statistical significance. This means that the terminal bouton numbers of fweDB25, pak3d02472/ fweDB56, Df(3R)pak3 larvae are significantly different than those of fweDB25/fweDB56, indicating that loss of pak3 increases the number of terminal boutons and enhances mutant flower synaptic phenotypes. Figure 2 graphically illustrates this relationship.
Figure 2. Simultaneous pak3 and flower mutations significantly increase terminal bouton numbers: the graph displays the terminal boutons for each genotype. The terminal bouton number of fweDB25, pak3d02472 / fweDB56, Df(3R)pak3 larvae is increased from that of both of the controls. The p-value calculated from Student’s t-test between fweDB25/fweDB56 and fweDB25, pak3d02472 / fweDB56, Df(3R)pak3 is .037, which is statistically significant. Statistical significance is indicated by the (*). For total bouton numbers, the p-value calculated from Student’s t-test between fweDB25/fweDB56 and fweDB25, pak3d02472/ fweDB56, Df(3R)pak3 is 0.054, which is barely above the significance cutoff of 0.05; counting more larvae should increase the statistical power. Thus, it is likely that deleting both pak3 and flower results in different total bouton counts than just deleting flower. Figure 3 offers a graphical representation of terminal bouton numbers for each genotype. Figure 4 provides representative images of NMJ synapses for each genotype. Collectively, the differences in total and terminal bouton numbers indicate that loss of pak3 enhances synaptic phenotypes exhibited by the loss of flower, specifically increasing the extra terminal, and likely total, boutons. Thus, pak3 and flower functionally interact in a positive and synergistic manner, in which the function of both genes is to aid in the proper formation and development of synaptic boutons.
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Figure 3. Simultaneous pak3 and flower mutations likely affect total bouton numbers: similar to terminal boutons, the average total bouton number of fweDB25, pak3d02472 / fweDB56, Df(3R)pak3 larvae is increased from that of both of the controls. The p-value calculated from Student’s t-test between fweDB25/ fweDB56 and fweDB25, pak3d02472 / fweDB56, Df(3R)pak3 is 0.054, which is barely above the significance cutoff of 0.05. Independent yet interrelated molecular pathways Since loss of pak3 enhances the extra boutons created by loss of flower and increases the severity of the phenotype, pak3 and flower are likely parts of two independent bouton-forming pathways. Removing any one component of a single pathway would disrupt it and likely render the pathway useless (so removing another part would have no additional affect since the pathway has already been disrupted), whereas with multiple signaling pathways, removing parts of both bouton development pathways would cause defects in both, leading to a more severe phenotype than if only one had been disrupted. Additionally, we know that loss of pak3 has little or no effect on bouton number on its own. However, pak3 is a necessary component in bouton formation and may be the only actin-remodeling part of the bouton formation process [15]. Since pak3 is an essential part of the pathway, we would expect its deletion to have a significant impact on the biochemical pathway. However, seeing as loss of pak3 alone has minimal effects on bouton structure, another molecule (likely a molecule that is part of the flower bouton formation pathway) may be able to take over the function of pak3. But, when both pak3 and flower are mutated, both bouton formation pathways are disabled, and there is nothing capable of taking over the actin remodeling function of pak3; as a result, the double-mutant phenotype becomes more severe than that of either the pak3 or flower mutation alone. Thus, although the pak3 and flower pathways leading to bouton formation are separate biochemical pathways, they are highly interrelated and interact with each other in order
Biology and Chemistry Research to ensure proper bouton formation and synaptic development.
pak3 NMJ, with the neuron stained with anti-HRP. Like wild-type NMJs, pak3d02472/Df(3R)pak3 NMJs are fairly simple, with relatively few branches (affecting terminal bouton count), few total boutons, and largesized boutons. This specific synaptic structure is considered “normal” and allows for proper functioning of the synapses. flower – As displayed in the image, the fweDB25/fweDB56 NMJ is much more involved than the WCS or pak3 NMJs, and includes more total and terminal boutons, as well as a highly branched and clustered structure. The individual boutons are also much smaller than normal. Together, these factors cause an abnormal synapse structure, leading to greatly compromised synaptic function. flower, pak3 – In this image, representing fweDB25, pak3d02472/fweDB56, Df(3R)pak3 mutants, the phenotype seen in flower mutants is significantly increased, with even more total and terminal boutons. A main axonal branch can be seen (going from the bottom left corner to the top right corner), with many smaller branches splitting off from the main branch. Many of these boutons appear even smaller than those at fweDB25/fweDB56 NMJs. Collectively, these characteristics provide strong evidence that loss of pak3 enhances flower mutant synaptic phenotypes.
Discussion
Figure 4. flower, pak3 double mutant NMJs display altered synaptic morphologies: wild type – this is a picture of a typical muscle 4 NMJ synapse in a wild type fly larva. The glia are labeled in green (gliotactin-Gal4, UAS-mCD8GFP) and the neurons are labeled in red (anti-HRP). The red circles are the synaptic boutons, specialized structures at the ends of axons, from which neurotransmitter is released onto the next cell (here, the muscle). This image provides several important insights. First, it shows the close interaction between glia and neurons, indicated by how proximal their positions are to each other. Secondly, it provides a reference for what “normal” NMJ synapses should look like. Additionally, it allows for a visual comparison between wild type and pak3 (pak3d02472/Df(3R)pak3) NMJs and shows that there is very little difference between the two. pak3 – This is a representative image of a pak3d02472/Df(3R)
From this investigation, we have determined that pak3, a primarily glial expressed gene that is involved in regulating actin projections, interacts synergistically with flower through separate yet connected pathways in order to regulate synapse structure and function, and that simultaneous deletion of both genes results in severe synaptic defects. This is important because it provides deeper insight into the function of pak3, a relatively unknown gene, illustrating that pak3 plays an important role in synapse development and regulation, and is not just spastin-specific. Additionally, although this study looks at the NMJs of fruit flies, the family of p21-activated kinases is evolutionarily conserved, and a similar gene to pak3 exists in vertebrates, including humans [23]. Thus, this study not only elucidates the molecular relationship between two genes, but also how those genes could potentially interact in human nervous systems. Another important connection is the relationship between pak3 interactions with flower and pak3 interactions with spastin. Even though both spastin and flower are neuronal genes that affect synapse structure and function, their relationships to pak3 are completely opposite – whereas loss of pak3 suppresses the extra boutons created by loss of spastin [19], loss of pak3 enhances the extra boutons created by loss of flower. This difference is noteworthy because it signifies that perhaps there are many bouton-forming pathways used by neurons, and pak3 happens to interact antagonistically with spastin (at least in bouton formation) and synergistically with Volume 3 | 2013-2014 | 59
Biology and Chemistry Research flower. Figures 5 and 6 provide a visual illustration of the proposed interactions between pak3, flower, and spastin at NMJs. Another aspect to consider is the site of action of pak3. Although it is primarily expressed in glia, pak3 is also expressed in a few neurons, albeit in a limited manner [15]. Thus, we must take into consideration the fact that some of the results we have observed could, in fact, be due to neuron-driven pak3 expression instead of glial-driven pak3 expression, as we have postulated. However, since pak3 is predominantly expressed in glia, and glia interact with neurons very closely (Figure 4), the synaptic phenotypes ensuing from pak3 deletion are likely a result of glialdriven pak3. Nevertheless, in order to test the possible discrepancies arising from glial versus neuronal-driven pak3 expression, it may be possible to delete pak3 only in certain cell types and compare the phenotypic results. Tissue-specific knockdown of pak3 in only neurons or in only glia, using RNAi, should reveal which can phenocopy the genetic interaction of the mutant alleles. This would allow us to learn more about the site of action of pak3, as well as reveal more information about the function of pak3 in cell types other than glia. For example, in what particular glia and neurons at what stages in development is pak3 being expressed?
Figure 5. A model of synaptic development at the NMJ: this model provides a visual illustration of the interactions between pak3, flower, and spastin at a typical Drosophila muscle 4 NMJ. pak3 is thought to regulate actin projections in glia [14][15]; Flower is a trans-membrane protein that mediates Ca2+ uptake during endocytosis and exocytosis; and Spastin serves to sever microtubules to allow for proper axonal growth. Both spastin and flower are primarily expressed in the neuron, while pak3 is mainly expressed in glial cells surrounding the neuron [15][17][21]; however, pak3 interacts genetically with both spastin [19] and flower in order to regulate synaptic structure and function. More specifically, pak3 interacts antagonistically with spastin [19] while pak3 interacts synergistically with flower. Details have been simplified in this diagram in order to allow for more focused examination of pak3, flower, and spastin. However, there are many other molecules (including secondary messengers, protein kinases, and other signaling molecules) involved in the 60 | 2013-2014 | Volume 3
actual biochemical pathways leading to proper synaptic development. Figure is not drawn to scale. All in all, this investigation also provides support for the growing consensus in the scientific community about the role of glia in the nervous system. Although glial cells were originally viewed solely as “support� cells for neurons, more evidence nowadays leads us to believe that glia play a critical role in proper nervous functions. In this report, our results support the idea that pak3 in glia is very important for the regulation of synaptic structure and function at the NMJs, and that glia and neurons work together in order to ensure proper development of synapses.
Figure 6. A closer look at synaptic regulation: this illustration is zoomed in on an individual synaptic bouton, in order to more thoroughly model synaptic regulation by pak3, flower, and spastin. Pak3, Flower, and Spastin proteins are shown carrying out their cellular functions, though actual molecular processes have been greatly simplified. Additionally, this model more accurately displays the close relationship between glia and neurons, with filopodial actin projections extending from the glial cell to the synaptic bouton on the neuron. Although the actual mechanisms of interaction between glia and neurons is unknown, this proposed model would allow the pak3 biochemical pathway to physically interact with the flower and spastin pathways at synaptic boutons, leading to our observed phenotypes. Also, the interactions between pak3 and flower and pak3 and spastin may or may not be related; in this diagram all three proteins are shown together in order to provide a more comprehensive picture of the role of pak3 in synaptic regulation. Figure is not drawn to scale.
Future Directions The results of this investigation have also raised new questions to be answered in future studies. Perhaps other genetic combinations could be tested, to see if pak3 interacts with other genes besides flower and spastin. One potential target is dap160, a primarily neuronally expressed
Biology and Chemistry Research gene that scaffolds the periactive zone of synapses and is involved in vesicle endocytosis and synaptic growth [24][25]. Since dap160 has similar functions to flower, perhaps pak3 not only interacts with spastin and flower, but also with dap160 (and possibly with other synapseaffecting genes as well). Investigating these other possible interactions would provide us with greater insight about the breadth of pak3 functionality, as well as illustrate the specific relationships between pak3 and other genes. Another interesting extension would be to test the relationship between flower and spastin, particularly since they have opposite interactions with pak3 but qualitatively similar mutant phenotypes on their own (increased numbers of small synaptic boutons), to see how they would interact with each other. This would allow us to take a step back from specifically looking at pak3 and increase our overall understanding about the complex and involved nature of synapse development and regulation. Finally, it would be noteworthy to examine other glial pathways, besides the pak3 pathway, which are known to affect synaptic bouton formation. draper, a glial expressed gene, is involved in the engulfment of destabilized presynaptic boutons and the disposal of neuronal debris during synaptic development, and affects synaptic bouton numbers [11]. Preliminary data suggests that draper, similar to pak3, is able to suppress spastin [15]. What is the effect of draper on flower though? Will draper interact synergistically with flower to regulate synaptic structure and function, like pak3, or antagonistically, like its interaction with spastin? Answering these fundamental questions would allow us to continue to expand our understanding of the role of glia in synaptic development and regulation. In summary, pak3 is a highly intriguing gene that appears to play a significant role in synapse development and displays great potential for future research and investigation.
Acknowledgments I gratefully thank Nina T. Sherwood for allowing me to work in her laboratory and utilize the resources available to conduct my research project. I also thank Emily Ozdowski, Chris Crowl, and Myra Halpin for their mentorship and support throughout my project. Thanks to Charlene Chen for her technical assistance with larval fillets and Esther Park for genetic recombination of mutant alleles. WORKS CITED [1] Collins, C. A., and DiAntonio, A. 2007. Synaptic development: insights from Drosophila. Curr. Op. Neuro. 17, 35-42. [2] Choquet, D., and Triller, A. 2013. The dynamic synapse. Neuron 80, 691-703. [3] Venken, K.J.T., and Bellen, H.J. 2005. Emerging technologies for gene manipulation in Drosophila melanogaster. Nat. Rev. Genet. 6, 167-178.
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