March 27, 2014
11:00 am to 12:00 pm EDT

Sponsored by

Symposium Course Description:

Studying Cellular Processes by high-throughput electroporation of primary cells

High-throughput screening of cancer cell lines is a powerful technique to identify lead compounds for drug discovery. Many advances have been made over recent years to facilitate cell-based screening and these include improvements of cell culture methods, automated handling and image acquisition. Classical screening approaches have used transformed cell lines such as HeLa or human embryonic kidney HEK293 cells in monolayer cultures. It has now become evident that these model systems do not accurately reflect human physiology, which may also explain a high attrition rate in current drug discovery programs. A paradigm shift has been proposed whereby performing biomedical experiments in primary cells or more complex three-dimensional environment will enable a better understanding of human cellular behavior and provide a higher success rate for the development of drugs. We are using the power of high-throughput screening approaches in order to understand the fundamental regulation of cellular processes such as mitogenic signal transduction and autophagy. More recently, we are developing novel approaches to high-throughput screening, using primary cell cultures and three-dimensional model systems in order to get a physiologically more relevant view on cellular processes. In this talk, I will give an overview of how we use these approaches to study the molecular regulation of autophagy.

Mechanistic insights into axonal mRNA transport

Mature brain function relies on a precisely sculptured neuronal network, which forms when growth cones at the tips of extending axons are guided to precise cellular targets by extracellular cues. After establishment, the maintenance of such a network is achieved through spatial restriction in protein expression. The response to guidance cues and restricted protein expression rely in part on the local translation of mRNAs transported within axons and growth cones. However, only a small number of axonal transcripts have been characterized and the mechanisms that regulate their localization and translation are largely unknown. Primary cultures of sympathetic neurons depend on Nerve Growth Factor (NGF) for survival and differentiation and are an ideal model to study mRNA transport and local translation during neuronal development as they grow very long axons in vitro. We grow primary sympathetic neurons in compartmentalized chambers to purify axonal and cell bodies mRNAs. We then combine imaging and sophisticated molecular biology techniques, such as SAGE and RACE, to characterize axonal mRNAs and their regulatory sequences. I will illustrate how using these approaches we have been able to identify a novel regulatory element that directs mRNA localization and translation in axons in response to NGF.

Learning Objective

• Understand cellular regulation of axonal mRNA transport
• Gain mechanistic insights into cellular processes in neurons
• Learn about phenotypic screening approaches for autophagy
• Learn about phenotypic screening in primary cells and neurons
• Gain insights into cell biology and molecular biology combinatorial approaches to investigations in primary neurons

Speaker Information

Robin Ketteler, PhD

Group Leader, MRC LMCB, University College London

Robin Ketteler studied biochemistry at the Free University Berlin and did his PhD at the Max-Planck-Institute for Immunobiology. After post-doctoral work at the Massachusetts General Hospital, Boston, in the lab of Brian Seed, Robin established the Translational Research Resource Centre at MRC LMCB in London in 2009. This academic high-throughput screening centre facilitates siRNA, cDNA and chemical screening in primary cells and cell lines. Main research interests focus on autophagy, protein trafficking, mitogenic signaling and virus infection.

Catia Andreassi, PhD

Senior Scientist, MRC LMCB, University College London

Catia Andreassi is a Senior Research Associate in Antonella Riccio lab at the MRC LMCB, University College London (UK). She received her MS in Biological Sciences from University of Pisa (Italy) and her PhD in Clinical Pathology from Catholic University in Rome (Italy). Much of Catia’s work has been focused on understanding the role of mRNA metabolism in brain development and disease. Initially, she studied the pathophysiology of Spinal Muscular Atrophy, a motor neuron disease where mutations in SMN, a ubiquitously expressed protein involved in several aspects of RNA metabolism, causes a neuronal specific phenotype. In 2005 she joined the Riccio lab where she has been leading a project aimed at investigating the role of Nerve Growth Factor in mRNA localization in axons of developing sympathetic neurons.

Cost: No cost