Correlative SXT and super resolution imaging to investigate cilia disease mechanisms in an animal model

Supervisor Organisation PhD Awarding Entity: phd location
University College Dublin
University College Dublin
University College Dublin, Ireland

Research Focus

This project will employ soft X-ray technology and super-resolution microscopy to image disease proteins within the primary cilium cellular organelle. Current CLEM methods are cumbersome and technically demanding, and very few studies have probed how cilia disease proteins are arranged relative to underlying structure, especially in metazoans.

This project will investigate combined cryo-SXT, FIBSEM and cryo-SIM approaches to image fluorescent protein reporters in a leading animal model (C. elegans nematode) of human cilia disease, focussing on highly conserved pathways essential for cilium formation and function.

The project will develop new CLEM methods for possible roll out to other tissue and animal model contexts, and provide insight of disease mechanisms based on subciliary compartmentalisation of associated proteins.

Methodology

High pressure and plunge freezing methods will be implemented for preparing 20 micron thick slices of the nematode nose that contain primary cilia. Optimized techniques for correlative fluorescence/substructure imaging will be developed in collaboration with CLEXM partners; soft X-ray imaging will be performed with SiriusXT whose prototype benchtop microscope system is housed in UCD. Using optimised methodology, correlative imaging will be conducted on nematodes expressing endogenous (knock-in) fluorescence-tagged ciliary proteins.

Aim 1

Develop optimised methodologies for preparing and imaging nematode samples.

Aim 2

Determine nanoscale spatial distributions of fluorescence-tagged ciliary disease proteins, correlated with underlying subcellular structure

Aim 3

Assess how disease mutations impact correlated spatial distributions of fluorescence-tagged ciliary proteins.

Pictures Attached

The 1 mm long C. elegans animal model, and fluorescence and electron microscopy images of cilia in axial and radial orientations.

PhD Researcher

Name: Jakub Krstev 

University: University College Dublin

Supervisor’s Name: Dr. Oliver Blacque

Jakub Krstev completed his Bachelor’s and Master’s degrees in Biotechnology at the University of Aberdeen. During his bachelor’s project, he became enamoured with cell culture, CRISPR and microscopy at the lab of Dr. Lionikas generating a POU3F4 knockout cell line. During his Master’s project, he worked under Dr. Harrington at TauRx Pharmaceuticals, his first experience in drug discovery and assay development. After completing his degrees he briefly working at APS Biocontrol as a research laboratory technician, working on trials of a bacteriophage-based biopesticide, before he joined the Alessio Ciulli – Boehringer Ingelheim (AC-BI) collaboration at the University of Dundee working as an associate cell biologist. As part of his role at AC-BI he was involved in the screening and development of various cell-based assays for the characterization of novel PROTAC molecule efficacy as part of a rational, multi-disciplinary drug discovery program, liaising and coordinating closely with chemists and biophysiscists on the team to maximise the value of data generated from cell-based screens. He joined the CLEXM doctoral network to be able to develop his skills in microscopy for which he possessed a great passion since his Bachelor’s, but had a limited scope for in his previous positions, as well as the exciting prospect of working with the groundbreaking Sirius SXT microscope, pioneering the use of SXT for C. Elegans and ciliopathy disease models.

His research interests are broad in their scope, encompassing everything from CRISPR, complex cell culture models such as organoids, all the way to correlative and high content microscopy. Additionally he is interested in building collaborations with discovery groups in order to maximise the utility of the models I develop for the field at large.

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