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Molecular &
Cellular Physiology
Laboratory

exocytosis, endocrine cell biology, cell signalling...

Dynamin co-localises with vesicles, as demonstrated
with co-localisation of dynamin protein with NPY

Research Summary

Our research is focused on the regulation of cell communication. Specifically we are interested in identifying new molecules which regulate the release of neurotransmitters and hormones. As well as this, we have a significant focus on how cell signalling is altered in human health disorders such as Type 2 Diabetes and obesity. We utilise human primary cells, genetically modified mice and molecular biology approaches, enabling us to investigate the role of specific proteins from the whole animal, single cell and single vesicle level.

These studies are carried out using a number of approaches including physiological measurements of exocytosis from single cells with carbon fibre amperometry, or by whole cell capacitance measurements and by the measurement of ion channel currents using patch clamp electrophysiology. We also carry out transfection of cells with GFP-tagged mutant constructs, immunohistochemistry, live cell imaging, real time RT-PCR, measurement of mitochondrial activity and culturing of primary cells and cell lines. These are complimented with whole animal in vivo studies including metabolic profiling of transgenic and knockout mouse lines.

Research Projects

Enterochromaffin (EC) cells are enteroendocrine cells lining the gut wall that supply ~95% of our total body serotonin (5-HT). They constitute half of all enteroendocrine cells, a group of cells that collectively represent the largest endocrine tissue in the body. This 5-HT plays a variety of paracrine and endocrine functions but the function of EC cells is poorly understood. We have developed methods to isolate single primary EC cells in culture with high purity from humans and rodents. This enables us to study the mechanisms that control 5-HT release from single EC cells. We have identified that these cells release 5-HT via mechanisms which are vastly different from other endocrine cells. We are currently identifying the mechanisms that control this release in human, mouse and guinea-pig EC cells and how these change in human health disorders such as Type 2 diabetes and obesity.

One of the core research areas in our group is identifying novel regulators of exocytosis, the process by which secretory vesicles fuse with the plasma membrane to release their contents. We use single cell electrochemistry and electrophysiology techniques to analyse the release of neurotransmitters and hormones at the level of single vesicle release. Current projects revolve around identifying how specific proteins regulate cell signalling through exocytosis, endocytosis and vesicle trafficking.

We are currently testing various mutant mouse models to try and identify novel genes/proteins in the regulation of pancreatic function and insulin secretion. We have identified that the RCAN1 protein is a novel regulator of beta cell function and insulin secretion in Type 2 diabetes and are currently trying to understand the mechanisms by which this regulation occurs.

Selected Publications

Peiris H, Duffield MD, Fadista J, Jessup CF, Kashmir V, Genders AJ, McGee SL, Martin AM, Saiedi M, Morton N, Carter R, Cousin MA, Kokotos AC, Oskolkov N, Volkov P, Hough TA, Fisher EM, Tybulewicz VL, Busciglio J, Coskun PE, Becker A, Belichenko PV, Mobley WC, Ryan MT, Chan JY, Laybutt DR, Coates PT, Yang S, Ling C, Groop L, Pritchard MA, Keating DJ (2016) A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes. PLoS Genet, 12(5):e1006033

 

Rogers GB, Keating DJ, Young RL, Wong ML, Licinio J, Wesselingh S (2016) From gut dysbiosis to altered brain function and mental illness: mechanisms and pathways. Molecular Psychiatry, 21(6):738-48

 

Young RL, Lumsden AL, Keating DJ (2015) Gut Serotonin Is a Regulator of Obesity and Metabolism. Gastroenterology, 149(1):253-5

 

Jackson J, Papadopulos A, Meunier FA, McCluskey A, Robinson PJ, Keating DJ (2015) Small molecules demonstrate the role of dynamin as a bi-directional regulator of the exocytosis fusion pore and vesicle release. Molecular Psychiatry, 20(7):810-9

 

Papadopulos A, Gomez GA, Martin S, Keating DJ, Jackson J, Gormal RS, Yap AS & Meunier FA (2015) Activity driven relaxation of the cortical acto-myosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking. Nature Communications, 6:6297

 

Kumar R, Corbett MA, Smith NJC, Jolly LA, Tan C, Keating DJ, Duffield MD, Utsumi T, Moriya K, Smith KR, Hoischen A, Abbott K, Harbord MG, Compton AG, Woenig JA, Arts P, Kwint M, Wieskamp N, Gijsen S, Veltman JA, Bahlo M, Gleeson JG, Haan E and Gecz J (2015) Homozygous mutation of STXBP5L explains an autosomal recessive infantile-onset neurodegenerative disorder. Human Molecular Genetics, 4(7):2000-10

 

Mackenzie KD, Duffield MD, Peiris H, Phillips L, Zanin MP, Teo EH, Zhou X-F and Keating DJ (2014) Huntingtin-associated protein 1 regulates exocytosis, vesicle docking, readily releasable pool size and fusion pore stability in mouse chromaffin cells. Journal of Physiology, 592(7):1505-18

 

Raghupathi R, Duffield MD, Zelkas L, Meedeniya A, Brookes SJH, Sia TC, Wattchow DA, Spencer NJ & Keating DJ (2013) Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells. Journal of Physiology, 591(Pt 23):5959-75

 

Zanin MP, Mackenzie KD, Peiris H, Pritchard MA, Keating DJ (2013) RCAN1 regulates vesicle recycling and quantal release kinetics via effects on calcineurin activity. Journal of Neurochemistry, 124(3):290-9

 

Peiris H, Raghupathi R, Jessup CF, Zanin MP, Mohanasundaram D, Mackenzie KD, Chataway T, Clarke JN, Brealey J, Coates PT, Pritchard MA, Keating DJ (2012) Increased expression of the glucose-responsive gene, RCAN1, causes hypoinsulinemia, β-cell dysfunction, and diabetes. Endocrinology, 153(11):5212-21

 

Zanin MP, Phillips L, Mackenzie KD, Keating DJ (2011) Aging differentially affects multiple aspects of vesicle fusion kinetics. PLoS One, 6(11):e27820

 

Wen PJ, Osborne SL, Zanin M, Low PC, Wang HT, Schoenwaelder SM, Jackson SP, Wedlich-Söldner R, Vanhaesebroeck B, Keating DJ, Meunier FA (2011) Phosphatidylinositol- (4,5)bisphosphate coordinates actin-mediated mobilization and translocation of secretory vesicles to the plasma membrane of chromaffin cells. Nature Communications, 2:491

 

Jentsch TJ, Maritzen T, Keating DJ and Thevenod F (2010) ClC-3 – a granular anion transporter involved in insulin secretion? Cell Metabolism, 12(4):307-8

 

Keating DJ and Spencer NJ (2010) Real time recordings of 5-HT release from enterochromaffin (EC) cells reveals their role in the generation and propagation of colonic migrating motor complexes. Gastroenterology, 138:659-70

 

Pfeffer, C, Stein, V, Keating DJ, Maier H, Rinke I, Rudhard Y, Hentschke M, Rune G, Jentsch TJ, Hubner CA (2009) NKCC1-Dependent GABAergic Excitation Drives Synaptic Network Maturation During Early Hippocampal Development. Journal of Neuroscience, 29:3419-30

 

Maritzen T, Keating DJ, Neagoe I, Zdebik AA, Jentsch TJ (2008) Role of the vesicular chloride transporter ClC-3 in neuroendocrine tissue. Journal of Neuroscience, 28:10587-98

 

Yu Y, Chu P, Bowser DN, Keating DJ, Harper I, Tkalcevic J, Finkelstein DI, Pritchard MA (2008) Mice deficient for the Down syndrome-related gene Itsn1 exhibit vesicle trafficking abnormalities. Human Molecular Genetics, 17:3281-90

 

Keating DJ, Dubach D, Zanin MP, Yu Y, Martin K, Zhao YF, Chen C, Porta S, Arbonés ML, Mittaz L, Pritchard MA (2008) DSCR1/RCAN1 regulates vesicle exocytosis and fusion pore kinetics: implications for Down syndrome and Alzheimer's disease. Human Molecular Genetics, 17:1020-30

 

Boettger T, Rust MB, Maier H, Seidenbecher T, Schweizer M, Keating DJ, Faulhaber J, Ehmke H, Pfeffer C, Scheel O, Lemcke B, Horst J, Leuwer R, Pape H-C, Völkl H, Hübner CA, Jentsch TJ (2003) Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold. EMBO Journal, 22: 5422-5434

 


Investigator

  • Damien Keating, PhD, Professor, NHMRC Career Development Fellow (Level 2)

Support Staff

  • Amanda Lumsden, PhD, Research Associate

Students

  • Minh-Son To, combined BMBS/PhD Student

  • Alyce Martin, PhD Student

  • Emily Sun, PhD Student

  • Helen Dockrell, PhD Student

  • Salha Alshumrani, Masters Student

    Essra Yagub Aloyouni, Masters Student


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