Endosomes sort transmembrane proteins (cargoes) internalised from the plasma membrane between degradation in lysosomes or for recycling back to the cell surface. In this way, endosomal sorting controls the cell surface proteome and thus regulates numerous cellular processes including cell signalling. Understanding this phenomenon at the molecular level is not a new question, but while decades of work led to the identification of numerous machineries involved, how they really “work” is unknown. This project capitalizes on protein design and novel microscopy to address this question in human cells.
A major bottleneck to unravel the molecular mechanisms of endosomal sorting is that conventional microscopes fail to image this phenomenon. This is because endosomes move too fast and in 3-dimensions. Compounding this problem is the need to image the sequence of recruitment of numerous machineries implicated in endosomal sorting. To solve this problem, we recently established multispectral microscopy with James Manton, whereby multiple overlapping fluorophores can be imaged simultaneously. By combining this technique with our oblique plane light-sheet microscope, we can achieve the unprecedented temporal resolution of one 8-colour 3D volume of the entire cell per second.
The successful applicant will apply this novel technique to the endosomal sorting problem. To label endosomal sorting machineries, they will combine CRISPR knock-ins with polycistronic plasmids. Conversely, cargo imaging will rely on pulse-chase with designed monovalent binders labelled with bright organic dyes. They will then develop custom image processing pipelines to quantitatively measure the sorting of cargoes within the endosomal system at the single molecule level. This project will provide the first quantitative characterization of endosomal sorting in mammalian cells. We will then measure how this is perturbed by deletion/depletion of sorting machineries to reach a molecular understanding of this process.
This project would suit biochemistry or biology students. Coding skills would be an advantage.
References
Healy MD & McNally KE & Butkovic et al., (2023) Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome Cell.
Wu K & Bai H et al. (2023) De novo design of modular peptide-binding proteins by superhelical matching Nature. 616, 581–589
Valm AM et al. (2017) Applying systems-level spectral imaging and analysis to reveal the organelle interactome Nature. 546, 162–167