Microscopy-based pooled screening to elucidate signal transduction events
Identifying all the genes that are involved in the various signal transduction pathways that drive cell functioning, including proliferation, differentiation and migration, has proven to be a formidable task. Pooled genetic screens can be used to speed up discovery, but such screens are only applicable to a few ‘static’ phenotypes, predominantly the viability of cells. We have recently devised a manner to circumvent this limitation and apply the pooled screening format to basically ANY phenotype that can be recognized by light microscopy. Thus genetic screens for cell morphology, differentiation, cytoskeletal alterations, signal transduction events and much, much more will now be in reach both in living and fixed cells.
The project has an attractive mix of molecular biology techniques, advanced live-cell microscopy, FRET and FLIM imaging, and automation of analysis by state of the art AI techniques. The candidate will be trained to apply these techniques by experts, visit associated labs in Europe for secondments, and apply the new knowledge to address outstanding questions on the temporal control of signaling pathways in cultured cells and 3d models.
You will be one of 12 PhD candidates collaborating in FLIMagin3d, a MARIE SKŁODOWSKA-CURIE ACTIONS Doctoral Network aimed to prepare young and talented scientists for the development of advanced optical technologies that appreciates all pertinent aspects such as data, platforms, complementary modalities and tools to enhance and enrich imaging in biomedical sciences. These advances will become the key elements in a new generation of smart and accessible imaging platforms that enrich our analyses of biology, disease, development and drug discovery in a wide range of applications.
We are looking for an enthusiastic and talented candidate with a masters degree in life sciences and proven interest in light microscopy, molecular techniques and image analysis. Knowledge of FRET and FLIM techniques is a pre. The candidate will be stationed in Amsterdam, and there is ample time to learn by visiting our associated labs in the Network. As international exchange is key to this project, we seek a candidate with roots outside the Netherlands.
The Jalink lab has a long history in cell biology, live-cell microscopy, FRET sensor development and development of microscope instruments, in close collaboration with leading microscope vendors. We are part of the EuroBioImaging flagship node for functional imaging (LCAM) and one of the founders of NL-BioImaging. For further information and to apply, please send email to email@example.com
You thought of an intriguing biological problem to tackle and wish to pursue this yourself? Design and make your own DNA constructs, put them in suitable cells, master the type of fluorescence microscopy you need and use (or perhaps write?) the software necessary to get to the answers, all in one rotation. Depending on your skills and motivation, an unusually broad spectrum of research techniques can be acquired in our group.
If you would like to do research in our group to get a better insight in biophysics and imaging techniques we always have a number of places for students. Depending on what you would like to learn, your own ‘research topic’ to tackle is embedded in one of the different ongoing research lines, a least in part. This enables us to maintain high quality guidance and support during your period over here. The NKI accepts second-rotation students for training. The institute provides excellent facilities and our lab is very well equipped to do biophysics. Taken together, these factors guarantee an excellent spot to do a rotation, as illustrated by the fact that work of our students frequently results in a publication. At the moment we only accommodate internships of 8-10 months.
Current projects for students:
Our group has a long history in developing biologically relevant FRET sensors, to measure for instance Estrogen Receptor functioning, phosphatidyl inositol 4,5 bisphosphate (PIP2) and cyclic AdenosineMonoPhosphate (cAMP). cAMP is an important second messenger that activates Protein Kinase A (PKA). Our lab recently published a new EPAC-based sensor to measure cAMP (Klarenbeek et al PlosOne 2015). We are currently making a cell line stably overexpressing the cAMP sensor. This cell line will be used to screen for proteins influencing cAMP levels by performing a genome wide knockdown by siRNAs/Cripr Cas or by using small molecule inhibitors. In the course of ~8 months, you will learn about fluorescent proteins, FRET, FLIM, large scale screening and the instrumentation you need for that. Automated image analysis will have to be developed to analyze these data. The image analysis will be used to identify new players in the cAMP signaling cascade. Finally, these new players will have to be verified.
Besides cAMP and PIP2 we are also interested in other signalling molecules like Inositol triPhosphate (IP3) and calcium. The activation of pathways using these signalling molecules is in most cases well described, however, the inactivation and/or desensitisation is less well understood. A current line of research, therefore, now comprehenses the elucidation of the inactivation and desensitisation mechanisms of this signalling pathway. Several cell lines will be transfected with FRET sensors to measure PIP2, IP3 and calcium. Furthermore components of the signalling cascade will be downregulated via siRNA/Cripr Cas or overexpressed to test if, and if so, how they influence signalling. Via live-cell imaging possible translocations and/or conformational changes as read out by FRET will be used.
In line with the cAMP sensor we are also interested in (local) induced effects of cAMP by adenylate cyclase. Recently a Photoactivatable Adenylate Cylcase (PAC) is published. This PAC is able to activate adenylate cyclase in distinct parts of the cell by activating it by laser light. This method allows us to test the effects of cAMP on distinct structures of a cell. Also other photoactivatable receptors are currently made or have been published recently, for instance photoactivatable G-Coupled receptors (paGPCR’s). By this method we are able to activate by laser light. Some paGPCR’s have been published, but others are still under construction and have to be fully characterized.
These developments constitute ideal student projects. In the course of ~8 months, you will learn about molecular cloning, fluorescent proteins, FRET and the instrumentation you need for that. You will create new, improved version(s) and extensively test them with state-of-the-art equipment. Depending on the progress made, you will also be able to apply your own FRET sensor to address questions embedded in one of the research lines in our lab. Because of the speed of developments, it is impossible to always present the newest ideas on this site; it is best to contact us and talk one-on-one about a project that would really excite you.
If you are interested, motivated and curious to know a bit more feel free to contact us !!!!