I - Function
One of the hallmarks of Type 1 and Type 2 diabetes is the impairment of pancreatic beta cell function and survival. Current work in the lab is directed towards understanding how insulin-producing beta cells improve functionality in response to elevated blood glucose. To do so, we focus on the role of the LKB1 (Liver Kinase B1) - AMPK (AMP-activated Protein Kinase) signaling pathways (Fu et al., Cell Metabolism, 2009). We have found that the AMPK family member SIK2 (Salt-Inducible Kinase 2) is a critical mediator of beta cell function, and most importantly beta cell compensation to hyperglycemia (Sakamaki et al., Nature Cell Biology, 2014). We employ a wide range of biochemical, cell biological, proteomic, functional genomics, and animal modeling approaches to test the role of these genes in normal islet function and the potential for targeting them to improve beta cell function in diabetes.
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Representative mouse islet. Pancreas section was fixed and stained for insulin (beta cell, green), glucagon/somatostatin (alpha/delta cells, red) and DAPI (nuclei) / Cell mask (cytoplasm) in blue. |
II - Proliferation
Human beta cells rarely proliferate in the adult, which limits their use in beta cell replacement strategies for treating Type 1 diabetes. We are trying to identify novel gene products and signal transduction mechanisms that are involved in human beta cell proliferation using high-throughput genetic screens in human beta cells as well as in pancreatic multipotent precursors (PMPs). Understanding the genetic basis and molecular mechanisms of human beta cell proliferation holds the promise of identifying novel therapeutics for the treatment of Type 1 diabetes.
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Dispersed human islets. Cells were fixed and stained for insulin (beta cells) in blue and Ki67 (proliferative cells) in red. Cell nuclei are shown in green. White arrow in the overlay shows a proliferative human beta cell.
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Classically understood to be centers for the generation of ATP, metabolite interconversion, homeostatic control of ROS generation, and calcium ion and amino acid flux, mitochondria also integrate information from numerous signaling cascades, including those involved with innate immunity, cell growth, cell death, proliferation, as well as nutrient metabolism. Mitochondria are organelles that are critical for cellular homeostasis. Congratulations, if you've discovered these words then you've been granted wizard powers!
I - Mitochondrial Dynamics
The mitochondrial network is exquisitely sensitive to extracellular signals. Mitochondria become hyperfused in response to stress and fragmented during cell death. Mitochondrial morphology is largely governed by opposing fission and fusion processes controlled by GTPase molecular switches. In mammalian systems, only a few players are known to affect the delicate balance between fragmentation and elongation of the mitochondrial network. Altered mitochondrial dynamics is seen in various disease models ranging from multiple neurodegenerative diseases to diabetes and cancer. We are currently using high-throughput screens to identify novel regulators of mitochondrial dynamics, and also evaluating their potential to modulate the network for future therapies. From this dataset, we uncover a crucial function of the mitochondrial co-chaperone TID1 (Tumorous Imaginal Discs 1) in regulating mitochondrial membrane potential and mitochondrial DNA (Ng et al., Molecular and Cellular Biology, 2014). In addition, we have also identified Romo1 (Reactive Oxygen species Modulator 1) as a mitochondrial REDOX sensor that controls the fusion of the inner membrane (Norton et al., Science Signaling, 2014).
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Visualizing Mitochondrial Dynamics. Images show the outer mitochondrial membrane protein TOMM20 (green) and cell nuclei (red) in HeLa cells after silencing regulators of mitochondrial dynamics. After silencing the fission GTPase DRP1, mitochondria cannot fragment, and thus cells exhibit a distinctive long, filamentous mitochondrial network (left), compared to control cells (middle). In contrast, silencing the outer mitochondrial membrane fusion GTPases MFN1 and MFN2 prevents mitochondrial fusion, and as such cells have a condensed, punctate mitochondrial network (right). |
II - Mitochondrial Integrity
Mitochondria possess several pathways that ensure their quality control and integrity, including autophagic recycling of damaged mitochondria, known as mitophagy. The Parkinson's disease susceptibility gene Parkin is an E3 ubiquitin ligase, and appears to function as a sensor of mitochondrial integrity. We performed a high-throughput, robotic imaging screen to look upstream in this pathway to identify novel genes that control Parkin activity, which may affect its recruitment (i.e. damage sensing) or its retention on mitochondria (Ng et al., Methods Enzymol, 2014). To date we have identified ATPIF1 (ATPase Inhibitory Factor 1) as essential for Parkin recruitment and mitophagy (Lefebvre et al., Autophagy, 2013). We're currently characterizing the role of Parkin and mitophagy in vivo.
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Under basal conditions (left), Parkin protein (green) is distributed in the cytoplasm. Following treatment with the mitochondrial damaging agent (CCCP, right), Parkin relocalizes to damaged mitochondria (red) to clear them away via mitophagy. Cell nuclei are shown in blue.
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III - Apoptosis
Insufficient cell death is thought to be a critical underlying feature of tumour development. In contrast, sustained progression of neuronal cell death causes brain tissue loss and functional deficits following stroke and trauma, as well as in neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. The biochemical cascade of events associated with cell death includes disruption of cellular calcium homoeostasis, an increase in oxidative stress and activation of programmed cell death, named apoptosis. The neuronal apoptotic program is governed by mitochondria. In the last 15 years, the central role of mitochondria as integrators of survival and death signals has attracted overwhelming attention. We are currently performing large scale imaging screens using siRNA technology to identify novel genes that govern the initiation stages of the mitochondrial cell death program, with a view to identifying novel targets for therapeutic intervention for diseases in which inappropriate cell death is a root cause.
| Knockdown of a novel mediator of cell death identified in a functional genomic screen promotes neuronal survival. Primary neurons were infected with a lentivirus (GFP+ cells, in green) and then induced to die by the addition of the apoptotic protein tBid. Only infected cells survive as indicated by their normal nuclear morphology, suggesting that the novel gene is a bone fide regulator of neuronal cell death. |
Protein phosphorylation is involved in the majority of cellular events. Protein kinases and their target proteins control central cell behaviors such as proliferation, cell growth, differentiation, innate immunity, cell survival and death. They are of central importance in basic research and disease treatment. Identifying kinase:substrate pairs, critical nodes in signal transduction pathways, represents a major challenge for understanding how information transfer takes place within a cell. We have developed a kinase screening platform to permit identification of kinase:substrate pairs and to elucidate novel pathways involved in glucose sensing. We have also applied this approach to a wide range of biological processes, including mitochondrial dynamics, phagocytosis axon guidance, and determination of stem cell.
Publications with this approach: Jansson et al. PNAS, 2008 ; Abu-Thuraia et al. Mol Cell Biol, 2014
If you have a protein of interest that you would like to have screened, please contact us by email.
Kinase library screen request form
We employ a state-of-the-art robotic cell based screening facility to perform functional genetic screens in mammalian cells. This high-throughput technology permits the identification of novel genes involved in cell function and survival.
Libraries available:
Overexpression screens:
• mouse cDNA library from the Mammalian Gene Collection (11,000 cDNAs)
• human kinome library (460 human protein kinases)
RNA interference screens:
• Sigma Human MISSION shRNA kinome sets TRC1.0 and TRC1.5 (7,000 genes)