Despite how rigorous the research has been in respect to the biochemistry and compartmentalization capabilities of CTPsyn within D. melanogaster, the finer details of its regulation remain elusive. The fly is known to carry a relatively high-percentage of microRNA (miRNA) genes, disproportionate to its small genome size. It is therefore natural to suspect that CTPsyn, like many other genes, fall under the regulatory effects of these small but potent RNAs. We took advantage of readily available stocks of driver-GAL4 and UAS-miRX lines to overexpress 123 miRNAs, and thusly screen each one for the effects its excessive production would confer upon the morphology of ovarian cytoophidia. Prior to rigorous screening, however, a control phenotype was established specifically for each driver, as we realized that the ectopic expression of GAL4 was already sufficient in presenting changes unto cytoophidia. By comparing the outcomes generated by follicle-cell drivers (FCDs) against both germline and ubiquitous Act5c drivers, too, we showed that the regulation of CTPsyn filamentation in nurse cells may be non-autonomous, as the process appeared to be dictated by cell-to-cell communication (CCC) between them and the surrounding follicle cells.
Nine, six, and three each of cytoophidia elongating (CytEl), shortening (CytSh), and polarity-distorting (CytPol) miRNAs were subsequently identified. Of these, only two each of CytEl and CytSh were deemed powerful enough to be designated prime candidates. In silico miRNA target prediction upon the 3’UTR region of CTPsynIsoC, i.e. the only cytoophidia-forming isoform of dmeCTPSyn, in fact showed that two candidates, namely CytSh-miR-932 and CytEl-miR-975, may explicitly bind to the mRNA. Disappointingly, in vitro assays utilizing overexpression constructs to the region instead conveyed that none actually do. For a miRNA so well known for its imperativeness towards cell survival such as CytSh-miRNA miR-190, the resultant shortening of cytoophidia is more likely symptomatic of the dysregulation wreaked upon the cell by miR-190 hyperactivity. Two factors further cement the imperative nature of this miRNA: one is its knockout-lethal nature, as seen in vitro in death curve assays, where cells transfected with sponge-constructs die quickly at relatively low plasmid dosages. Another is its comparatively higher endogenous expression levels over that of the other three prime candidates, as established by sensor-based assays. However, none of miR-190’s most probable in silico predicted targets were convincingly linkable to CTPsyn. This too is true for CytEl-miR-975’s predicted targets.Conversely, plausible connections to cytoophidia were demonstrably shown through qPCR fluctuations of specific targets predicted for either CytSh-miR-932 or CytEl-miR-1014. The expression of fitm was downregulated in miR-932-overexpressing ovaries. The gene has known functions in compartmentalization of organelles and their localization within the cell. If its protein product is indeed involved in either of these events for CTPsyn cytoophidia, then its reduced levels may have disabled proper tethering, leading to the filaments’ truncated appearance. MiR-1014, meanwhile, directly downregulates CdsA, a direct consumer of CTP nucleotides at the advent of phospholipid biosynthesis. We reasoned that as its reduced mRNA levels translates to lower CdsA-enzyme activity, the resultant environment of CTP surplus inactivates CTPsyn molecules, which go on to polymerize and thereby materializing as longer cytoophidia.
A more extreme hypothesis derived from these two outcomes is that the fat metabolism pathway is heavily interwoven with the formation of cytoophidia, without intrinsically altering CTPsynIsoC levels. We believe that a small free-flowing organelles called lipid droplets (LDs) in particular, is explicitly influential to the filamentation process. Whereas CdsA is required to initiate fat production, fitm stores the produced fat into LDs. CdsA-knockdown mimics starvation, a situation which increases LD numbers, whilst fitm-knockdown mimics fat availability and slows LD production down. In miR-1014-overexpressing ovaries, cytoophidia is elongated concurrently to lowered CdsA levels; in miR-932-overexpressing ovaries, the structure is instead shortened concurrently to lowered fitm levels. Viewed together as a whole, these connections may allude to there being a physical relationship between LDs and CTPsyn as the protein undergoes filamentation.
Could LDs then be causal to the observed non-autonomy in cytoophidia formation within follicle-cell sheathed nurse cells? Though certainly feasible, justifying the ‘how’ is more complex. We have already nominated two molecules which are most likely to exhibit intercellular mobility i.e. the overexpressed miRNA itself, or CTP nucleotides. Where FCDs were utilized to drive overexpression, we had believed that cytoophidia-altering information may be conveyed from follicle cells to nurse cells through either the movement of miRNA or CTP acting as small signalling molecules. This occurs passively through the channels found interring across the membranes of neighbouring, in-contact cells. LDs tend to be involved in more dynamic events. They act as hubs for fatty-acid trafficking, both within and beyond the lipid-rich boundaries of a single cell. If CCC is to be instigated by LDs instead of miRNA and/or CTP, then the process would not take place through such passive means; rather, it would require the coordinated integration of LDs into the phospholipid bilayer and subsequent exportation of fat-rich globules to nearby cells with lower accumulative levels of LDs. However, non-autonomy does not extend both ways: follicle cell cytoophidia were not affected by the changes proffered by nos-GAL4-driven overexpression upon the macro-cytoophidia of connected nurse cells. As LD-assisted trafficking is a two-way street, it does not support the one-way CCC model we had already established. It is therefore more probable that LD-mediated cytoophidia formation is in itself the by-product of miRNA and/or CTP mobility between adjoining cells.
As we approached the final stages of in vivo validation, another interesting artefact was uncovered in the increased frequencies of bulged testes recovered with the overexpression of either one of CytEl-miRNA candidates. CTPsynIsoC-overexpression is known to induce the same phenotype. This made the discovery especially exciting, as it adds more support to the notion miR-975 and miR-1014 may have direct ties to CTPsyn regulation after all. It also enhances the long-acknowledged associations CTPsyn has had to cancer progression. Accordingly, qPCR for cancer-related genes confirms the upregulation of most in both Act5c>miR-975 and Act5c>miR-1014 testes. Of course, this does not automatically qualifies the phenotype as tumours. Nevertheless, in the case of miR-975, the idea that the miRNA is able to kick-start the cascade of events leading to tumorigenesis is supported by observations in their female counterparts. Apart from presenting similar genetic expression profiles with the select panel of oncogenes, a small proportion of egg chambers in ovaries overexpressing miR-975 also shoed hallmarks of germline-cell tumours (GCTs).
On the other hand, differences in expression profiles were glaringly obvious between the sexes of miR-1014-overexpressing flies. Females of the genotype additionally do not bear egg chambers with GCT-like traits. In fact, quite the contrary: immunostaining revealed nearly 25% of them actually undergoing apoptosis instead. The vastly polarizing phenotypes induced by miR-1014 overexpression in this case could hereby foreshadow that the effects of a particular miRNA could be as specific as to be influenced by sex. Are the affected factors here therefore the differential genes activated for the purpose of sex-determination? Or are the consequences of miR-1014 overexpression exacerbated by the microenvironment of the testes, which indubitably could not be mirrored within ovaries? Regardless, these outcomes display the usability of D. melanogaster testicular tissue as a model for studying cancer or cancer-like growth, with the additional advantages of being able to simultaneously account for sex and microenvironmental influences.
Overall, these three chapters were successful in ascertaining that miRNAs could indeed confer changes upon cytoophidia. Though it is unlikely that the CTPsyn mRNA is directly targeted by any of the identified candidates, the changes they induce are substantial enough to visibly alter cytoophidia morphology. Although all preliminary conclusions derived should be treated with caution, several questions arising from the outcomes within this dissertation are definitely worth addressing in the near future, such as: (a) whether the CCC taking place from follicle to nurse cell does indeed dictate cytoophidia formation; (b) whether LDs are physically associated with CTPsyn-cytoophidia; (c) whether CytEl-miRNAs qualify as onco-miRs; and (d) how integral sex and/or microenvironmental factors are to the whole picture.
General conclusion for molecular characterization of CTPsyn in Ae. aegypti and Ae. albopictus
In this study we comprehensively demonstrated the distribution and localization patterns of CTPsyn in Aedes tissue. Whilst largely absent from germline cells, its protein is a significant presence in tissues involved in osmoregulation, excretion, and overall hemolymph transport. Coincidentally, these are the same tissues which would successively ‘receive’ pathogens once a female mosquito has ingested tainted blood. For both Ae. aegypti and Ae. albopictus, high-levels of nuclear-CTPsyn (n-CTPsyn) was detected in Malphigian tubules, small intestines, and the rectum. More n-CTPsyn was found concentrated within the nuclei of membrane-lining cells of the midgut in Ae. albopictus. Though no incidence of cytoophidia sightings was recorded in any tissue, CTPsyn appear to aggregate into perinuclear aggregates in Malphigian tubules in a temporally-dependent manner. This trait was also observed upon blood-feeding, as was the quick build-up of CTPsyn within the nuclei of nurse cells in reactivated ovaries.
However, quantification of CTPsyn showed, for the most part, rather contrasting protein to transcript relationships in adult tissues. The apparent spike in CTPsyn protein in ovarioles of blood-fed females is countered by a downturn in levels of its mRNA, whereas males see relatively high-levels of CTPsyn, despite displaying almost entirely CTPsyn-devoid gonads. These paradoxes not only attune our expectations on the versatility of the protein’s functions within the cell, but also allude to the possibility that the accumulation of CTPsyn observed in nurse cells after blood-feeding was the result of cytoplasmic to nuclear translocation events, rather than induced upregulation in CTPsyn expression. RT-qPCR data nonetheless showed that cell cultures of either species express higher-than-species-average levels of CTPsyn. Upon treatment with DON, a glutamine analogue drug, its resultant proteins undergo filamentation to produce cytoophidia. This means that eventhough the structure cannot be formed in any tissue in vivo, the Aedes genus does have the means necessary to facilitate its formation, and that the conservation of this mechanism towards protein organization may bear some degree of importance throughout the course of organismal evolution. In the near future, we hope that with improved skills at tissue handling and immunostaining, additional knowledge such as CTPsyn distribution in embryos, larval and pupal tissues, as well as other adult tissues will be revealed. The generation of mosquito lines carrying a CTPsyn-reporter construct could also allow true visualization of the protein’s movement throughout the life cycle of the mosquito. If these are refined to be variant-specific, it would provide an even clearer picture of differential localization amongst isoforms as well as CTPsyn behaviour in general.
Based on the consistent association between the protein and hemolymph/fluid transport tissues, we suspect that CTPsyn functions and products are some of the host’s resources exploited upon by opportunistic pathogens for purposes serving their own replication and population growth, as they travel through these channels back to the salivary gland. A recommended continuation to this project should involve the knockout or attenuation of CTPsyn expression, achievable through either traditional genetic engineering, or cutting edge and brand-new CRISPR-Cas9 technology. This is to be followed by infection efficacy assays: negative effects of CTPsyn absence on pathogen progression should provide credibility to this theory, and ultimately indicate whether CTPsyn protein could be a novel candidate target in strategies for control of vector-borne pathogens.