CORDIS - EU research results

Innovation and application of genetically-encoded probes for correlated live-cell imaging and electron microscopy

Periodic Report Summary 2 - PROBES FOR CLEM (Innovation and application of genetically-encoded probes for correlated live-cell imaging and electron microscopy)

The aim of this project was to develop, validate and implement a probe for combined fluorescence live-Cell imaging and electron microscopy (CLEM) of proteins of interest. After the first reporting period, a proof-of-principle was achieved for live-cell imaging of the Golgi-complex using a single colour, followed by EM-analysis. In the second period, we achieved the combinatorial probe with multiple colours, multiple targets and the implementation in research projects as detailed below. Thus, during the project, we engineered and applied a genetically-encoded tag consisting of a fluorescent protein (highly suitable for live cell imaging) and a module to visualise proteins by electron microscopy at high resolution with high quality preservation of the ultrastructure to form Fluorescent indicator and (per)oxidase for precipitation with EM resolution (FLIPPER).

FLIPPER is genetically-encoded and combines advantages of FPs and HRP for high spatiotemporal imaging.

Initial work focussed on the construction of the combinatorial fusion and fusion to a target protein for specific subcellular targeting, which provided a solid and successful basis for realising the main goals. Thus, the proposed FLIPPER module has been constructed and has been applied for different imaging modalities. As a proof of principle, targeting to the Golgi-apparatus has been accomplished after reporting period 1. We validated FLIPPERs to target the Golgi apparatus, endoplasmic reticulum and the secretory route, both for live cell analysis and high resolution EM-imaging. Moreover, also multicolour, i.e. red and green, FLIPPERs are now available. The FLIPPERs are being implemented in divers (collaborative) effects, including examination of ER-stress, define precise protein localisation, Golgi morphology during stress.

We produced new genetically-encoded probes for CLEM in cell biological research. The added value of these probes over existing ones is that:
(1) the targeting is highly specific and;
(2) the genetic encoding allows for high quality preservation of the ultrastructure.

Moreover, since the probe is genuinely genetically-encoded, and easy to use (compare with the green fluorescent protein (GFP)), it can be readily made available to the entire research community in Cell Biology and the new tool is already being used in several (collaborative) projects as discussed above. Upon publication of our manuscript on FLIPPER, we expect an increase demand of other researchers, some of them already expressed their interest of applying this new tool in their research. In the meantime, the construction of a generic FLIPPER that can be used to monitor cytoplasmic proteins has been initiated, but we did not yet succeed. Future efforts will be made to engineer a cytoplasmic FLIPPER-like module.