Towards exploring protein clusters: determining stoichiometry and recruitment kinetics via single molecule microscopy

Tashev, Stanimir Asenov ORCID: 0000-0002-0119-4412 (2024). Towards exploring protein clusters: determining stoichiometry and recruitment kinetics via single molecule microscopy. University of Birmingham. Ph.D.

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Abstract

Cells are dynamic systems, in which proteins regularly form complexes of changing stoichiometry. However, molecular counting techniques are mainly performed on fixed cells, thus losing the temporal aspects of complex formation, which might have revealed additional information of underlying biological processes. This PhD thesis presents developments to the quantitative microscopy technique Counting by Photon Statistics (CoPS), contributing towards towards making the technique a live cell compatible method. The work involved constructing a microscopy setup with the ability to acquire antibunching data, as well as to support widefield modalities. The microscope characteristics were defined and CoPS measurements on it were validated. A molecular standard with consistent DNA-hybridisation kinetics, the rates of which were defined, was used to test new dynamic versions of CoPS better suited for continuous data. However, photodestruction of the model system was observed with Cy5 labelling, which decreased when labelled with Atto 643. Experiments on cells were conducted to explore the challenges of in cellulo CoPS. The optimal imaging conditions were explored and CoPS experiments were conducted on a cellular molecular standard. The recruitment of the adaptor protein SLP-76 protein was also inspected as a dynamic system of protein recruitment, and the stoichiometry of its clusters was identified. A new technique for determination of the degree of labelling (DOL) was developed to facilitate the accuracy of these measurements. The limiting factor of live cell CoPS was determined to be the photophysics and photostability of the fluorophores. For the technique to work a further large study of different fluorophores and live cell buffers was identified as a direction of future experiments.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):