SystemsX.ch: CINA - The Proteomics Imaging Pipeline

The Center for Cellular Imaging and NanoAnalytics (C-CINA) is being established to pursue the goal of SystemsX.ch to acquire quantitative information on cells and organisms and to expand our understanding of biological systems. This Center will provide measuring and imaging techniques for nanoscale cell biology. Tools and methods will be developed and established to image single cells at nanometer scale resolution and to determine the proteome of single cells by high-throughput visual proteomics. Such tools will be available to the partners of SystemsX.ch, and will be constantly developed according to the needs of SystemsX.ch. Strong expertise in nanoscale science is essential for state-of-the art cellular nanoanalytics for systems biology: the Basel based National Center of Competence in Research (NCCR) Nanoscale Science is an excellent knowledge platform for such developments. The C-CINA will become a partner in this consortium. Moreover, the C-CINA will closely collaborate with the Department of BioSystem Science and Engineering (D-BSSE) of the ETH.

Electron microscopy (EM) is used to visualize supramolecular complexes, organelles and cells. Cryo-electron tomography of vitrified cells is currently the only method to assess the biological machinery involved in signal transmission and gene regulation in the cellular context, at nanos-cale resolution. This noninvasive three-dimensional imaging technique is a unique method to un-ravel the architecture of biological systems. To study the proteome of a single cell, however, we develop microfluidic devices that allow cells to be grown under reproducible conditions, perturbed, lysed, fractionated and deposited on the EM grid by a spotter. In collaboration with the Hierlemann group at the D-BSSE, we build these devices using micro-fluidics. The entire content of a cell can yield 500-5000 suitably diluted 10-pico-liter (pL) fractions, each one producing a 30-100 μm spot, which fits to one grid square. With one EM grid providing several hundred such grid squares, we will be able to sample the entire proteome of a cell with a handful of grids. The EM will image these spots in an automated fashion, and thereby read the pages of the book that represents the cell’s proteome. Since the microfluidic process will be fast, and will allow complexes to be immediately stabilized by appropriate cross-linkers, the native state of the nodes representing a signaling network has a high likelihood of being pre-served within a very short and well-defined time after perturbation. The resulting EM sample is sufficiently thin for a resolution of 1-2 nm to be achieved, thereby allowing complexes to be iden-tified based on their high-resolution projections using advanced pattern recognition algorithms. Gold-labeled antibodies or FAB fragments that produce distinct signals will be used to further enhance identification. Such samples can be freeze-dried and analyzed by the scanning trans-mission electron microscope (STEM), which delivers the mass of the deposited complexes. This bridge between mass and shape is key to linking mass spectrometry data and structural informa-tion, and will facilitate interpretation of large-scale proteomics analyses. We will develop this proteome imaging approach to full automation, thereby making it suitable for high throughput analyses. We also propose to combine it with mass spectrometry of cross-linked complexes in the 2 MDa range in collaboration with the Zenobi laboratory at the ETHZ, and with the multiarray opt-ical tweezers system developed in the Vogel laboratory at the EPFL.

Light optical microscopy (LM) has made tremendous progress in imaging of life cells, not only by the development of imaging modes and digital data acquisition, but also by the availability of chimera of fluorescent protein (FP) and specific marker proteins. To take advantage of these possibilities, we will design optical systems that allow vitrified samples to be observed by light microscopy for identification of suitable areas for electron tomography, as well as to monitor the fate of protein complexes during fractionation in the microfluidic system. Yet, since the routinely achieved resolution of LM is 300 nm and that of electron tomography 3 nm, ultrastructural analyses at 30 nm resolution of tissue volumes reaching (300 μm)3, is an important achievement of serial block face imaging. This technique is also established at the C-CINA in collaboration with the FMI.

The C-CINA will acquire multi-scale and multi-modal information about cellular proteomes by using a heterogeneous array of instruments and methods. These data will be integrated with data from other methods within the SystemsX.ch network, such as mass spectra and genomic sequence data. The C-ISD node of SystemsX.ch is developing the openBIS biological database that allows scientists to manage and mine data from different disciplines that are connected in ontological hierarchies. The C-CINA will maintain the electron microscopy laboratory database EMEN2 that is developed by the group of Steve Ludtke in the Baylor College of Medicine, Houston, Texas. EMEN2 is specialized for the large data volumes and image processing workflows that EM work requires. We interface EMEN2 with the openBIS system, to make the EMEN2 functionality available to the openBIS database. This will provide scientists from SystemsX.ch with a coherent interface for data archiving, visualization, and mining of information about cellular proteomes.

In collaboration with Andreas Hierlemann (D-BSSE, Basel, ETHZ), Renato Zenobi (ETHZ), Marcus Textor (ETHZ), Horst Vogel (EPFL), Susan Gasser (FMI),Susan Paszkowski (UniL), Ruedi Aebersold (ETHZ), Ari Helenius (ETHZ), and Berd Rinn (C-ISD, D-BSSE).