The aim of this project is to set up an advanced imaging technique, Expansion Microscopy (ExM), to study localization of nuclear proteins, protein-protein and protein-nucleic acids interactions relevant for transcription regulation.
ExM is a sample preparation technique aimed to swell cells in order to enable or improve the identification of protein-protein interactions at nanoscale resolution in highly crowded environments such as nuclei. In this technique, fixed and immunostained cells or tissues are embedded in a hydrogel that expands upon addition of deionized water (Chen F, Tillberg PW, Boyden ES. Optical imaging. Expansion microscopy. Science. 2015 Jan 30;347(6221):543-8).
ExM, together with 3D multiparametric confocal acquisition and colocalization analysis, will be exploited to study the role of a recently identified protein complex (the Restrictor complex) involved in the control of extragenic transcription. Restrictor complex is composed by ZC3H4, a highly disordered RNA-binding protein and its binding partner WDR82, a Pol II-binding protein and limits the accumulation of non-coding RNAs, the uncontrolled transcription of which may promote harmful effects on nuclear organization and genome stability (Austenaa LMI, Piccolo V, Russo M, Prosperini E, Polletti S, Polizzese D, Ghisletti S, Barozzi I, Diaferia GR, Natoli G. A first exon termination checkpoint preferentially suppresses extragenic transcription. Nat Struct Mol Biol. 2021 Apr;28(4):337-346).
The setting up of the ExM technique (which includes the identification of the more suitable protocol and fluorophores to use, the identification of the proper acquisition settings and the image analysis using automated pipelines) will be of great value to shed light on the importance of a regulatory mechanism that controls extragenic transcription, as well as for other projects in which molecular interactions in the nucleus should be analysed. Moreover, the data obtained with ExM will be compared and corroborated with data obtained with the STORM (Stochastic Optical Reconstruction Microscopy) super-resolution technique (Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006 Oct;3(10):793-5).
The student will learn:
- Cell culture techniques
- Immunofluorescence labelling
- Expansion microscopy
- Wide-field and fluorescence confocal microscopy
- Super-resolution microscopy (STORM)
- Image analysis
The student should have successfully taken the exams of the master course within the prescribed period of time and have high marks (mean >28.5/30). He/She should be interested in understanding mechanisms of transcriptional control and in learning techniques related to sample preparation, optical imaging and image analysis.
The projects will last for 1 year and it will start as soon as a suitable candidate is identified.