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Linking reproductive behaviour and dense core granule biogenesis in secondary cells of the Drosophila male reproductive system

Reference	BB/N016300/1
Principal Investigator / Supervisor	Professor Clive Wilson
Co-Investigators / Co-Supervisors
Institution	University of Oxford
Department	Physiology Anatomy and Genetics
Funding type	Research
Value (£)	518,971
Status	Completed
Type	Research Grant
Start date	05/08/2016
End date	04/08/2019
Duration	36 months

Abstract

The mechanisms controlling regulated secretion by neurons, endocrine and exocrine cells ensure the appropriate molecules are released from dense core granules (DCGs) and DCGs replenished. We have developed the prostate-like secondary cell (SC) in flies as a new in vivo model to dissect the genetic processes controlling these events. By using the temperature-dependent GAL4/UAS/GAL80ts system, we can inducibly knock down candidate regulators of this process, while expressing fluorescently tagged membrane and DCG markers. We have pioneered pulse-chase, super-resolution and real-time microscopy techniques in this model to visualise the trafficking events involved in DCG biogenesis and release in intact living glands. A newly identified QF transcriptional driver now allows us to assess trafficking while genetically manipulating the neural circuits involved in mating. Using these approaches, we have shown that mating induces DCG release and autocrine BMP-dependent DCG replenishment. In addition, we have highlighted potential roles for vesicles inside DCG compartments and for intercompartmental cross-talk in DCG maturation. Our key objectives are now to employ our unique tools and approaches to: 1. characterise the mechanisms by which BMP signalling in SCs is controlled by mating and regulates DCC biogenesis in males at different ages; 2. test the roles of nanovesicles and the exchange of molecules between different compartments during DCG loading and condensation, and identify the functions of evolutionarily conserved regulators in these processes; 3. determine how neural circuits activated during mating control SC secretory activity. These studies should not only provide insights into the ways that mating induces subcellular changes in single cells to maintain secretory capacity and fertility, but will also reveal the detailed mechanisms that control DCG biogenesis, findings that are likely to be relevant to human diseases where secretion is defective.

Summary

All animals are formed from cells, each with their own functions, which work together to ensure that basic biological processes are kept in balance. To achieve this, neurons and many different cells within glands secrete signals in a controlled way. These signals instruct neighbouring or distant cells to change their behaviour when the normal balance is disturbed or when changes in the environment require the body to adjust accordingly. For example, beta cells in the pancreas secrete more insulin when blood sugar levels are raised, instructing other cells to take up the sugar and restore equilibrium, while nerve-like cells in the adrenal medulla release adrenaline in response to stress to prepare our bodies to fight or run. Cells involved in this regulated form of secretion share common features. They package the hormones or enzymes they release into special compartments, where the molecules are condensed into so-called dense core granules (DCGs) before secretion. Some mechanisms involved have been characterised in detail, but other aspects are poorly understood. For example, how do cells sense that they must rapidly replenish their DCGs after granule release? And how can this be co-ordinated with signals from the environment that alter secretion rates? While answering the first question relies on developing ways of testing how genes control the tiny compartments in which DCGs are formed, these compartments must also be studied in the whole animal to work out how they are affected by the environment. Since DCG control mechanisms go wrong in diseases like diabetes, where insulin secretion is defective, and cancer, where tumour cells signal inappropriately to normal cells around them, understanding how secretion is regulated has the potential to change the way we detect and treat these diseases. We are investigating this problem by studying special prostate-like cells called secondary cells (SCs) in the male reproductive system of the adult fruit fly. It is much easier to study the genetics behind secretion in flies than in animals like mice, and despite the fly's apparent simplicity, it shares remarkable similarities with humans. We have found that SCs have very large DCGs, some of which are released into seminal fluid each time a male fly mates. Mechanisms that control DCG formation in humans seem to be involved in making these compartments in SCs. But we have also found that molecules called BMPs, which are involved in sensing DCG release, instruct SCs to make new compartments, a mechanism that we think is conserved in mammals. In addition, we have been able to use a new type of super-resolution microscopy to watch the giant DCGs form in living glands for the first time, revealing other new features of this process. We now propose to work out the precise way in which BMPs control DCG formation and how brain activity during mating increases the BMP signal so more granules are made. In addition, we will test the importance of some of the new mechanisms for DCG control we have uncovered, such as the delivery of molecules to DCGs on nano-sized vesicles, which are also made by mammalian cells, but have not previously been linked to DCG formation. Overall, our proposed studies will use the unique biology of SCs and our ability to change their secretion using genetics and by mating flies to work out the ways in which different aspects of secretion are controlled in a living animal. Understanding the basic mechanisms involved may also help us to determine how they go wrong in other secreting cells: for example, in diseases like Type 2 diabetes, where faulty secretion leads to imbalance in the body's metabolic control systems, or cancer, where defective secretion can reprogramme normal cells to help tumour cells survive. We have already established collaborative links through our previous studies in flies to take this disease-led work forward as we gain new insights into the ways secretion is controlled from this project.

Impact Summary

In the previous section, we describe the wide-ranging implications of the proposed work and its potential impact on academic beneficiaries. Other possible areas of impact are: 1. Clinical Medicine Understanding regulated secretion mechanisms is potentially relevant to many major human diseases, including diabetes, cancer and neurodegenerative disorders. One collaborator on our recent CRUK-funded Programme grant, Adrian Harris, is a clinical oncologist and will provide clinical input via our monthly research meetings scheduled over the next five years. Previous interactions stemming from our fly studies have led to development of an antibody against the amino acid transporter PAT4 that detects upregulated expression in colorectal cancer patients with poor prognosis (Fan et al., 2015, Oncogene, in press). This antibody is being tested as a prostate cancer biomarker in collaboration with Freddie Hamdy and Aaron Leiblich, the clinical urologists whom I worked with to establish the fly SC model. Now in a surgical post, Leiblich will continue to work with me half-time to further cement these translational interactions. With these collaborations in place, we are therefore particularly well positioned to translate new discoveries from this proposal into studies of cancer cells that employ regulated secretory pathways. Through these connections, my post as an Oxford Medical Tutor and our membership of various focus groups in Oxford (BMP signalling, metabolism, cellular ageing, extracellular vesicles, Parkinson's) that are supported by clinicians and basic scientists in the Medical Sciences Division, we have links to other clinical areas, which could be exploited if we identify new mechanisms that might be relevant to diagnosis or treatment. 2. Pharmaceutical Industry and Biotechnology I am a member of ChemBio Hub, an organisation promoting dialogue between industry and Oxford academics. Through this, we have been approached by Ipsen about targeting specific secretory pathways in cancer. Although discussions are at an early stage, these interactions highlight the potential pharmaceutical interest in understanding specific secretory processes in disease, so that they can be selectively targeted or used for diagnosis. We will continue to actively promote industry conversations via ChemBio Hub and the contacts we make through it. It is conceivable that developing a better understanding of eukaryotic secretory mechanisms might ultimately inform approaches for large-scale production of bioactive proteins, which we would also pursue through this route. Finally, we have contacts with Luke Alphey (Oxitec, a company focusing on insect pest control), through which we can explore possibilities of using our findings, for example in improving competitiveness of male insects used in mass release strategies. 3. General Public and Schools Our workexemplifies how invertebrates can be used to undertake in vivo studies, which target fundamental problems that are ultimately relevant to human health, but are also of basic biological interest. The mechanisms that drive the conflict between sexes during reproduction fascinate the public, particularly since some of them may be conserved in humans. We recently applied to the Wellcome Trust for funds to support an art project linked to schools, involving imaging in my and other neighbouring labs; although this was not successful, we plan to pursue this further. In addition, our recent CRUK grant has opened up new opportunities via the CRUK Engagement Manager to engage the public with an interest in health science through talks, events and lab visits that we will take part in. I also give talks at schools and I am in discussions to organise a taster day at my College St. Hugh's for medicine and biomedical sciences, where students, particularly from schools that do not have strong links with Oxford, have the opportunity to visit and also see how academic science can impact on healthcare and industry.

Committee	Research Committee A (Animal disease, health and welfare)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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