The rainbow cell - enabling technologies for immunofluorescence of living cells

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre

Abstract

Background Current strategies to image individual portions of the cell either require genetic modification to introduce fluorescently-labelled proteins or fixing and permeabilisation of cells followed by immunofluorescent staining with antibodies. In this project, the student will develop technologies to effectively label membrane compartments in living cells without such genetic modification. The strategy will be to deliver fluorescently-labelled Adhirons into the cell using bacterial toxins as a delivery vehicle.

Objectives

1. Select Adhirons with selective binding to specific subcompartments (e.g. ERGIC-53, Man6p receptor);
2. Demonstrate on-target engagement of these Adhirons in cells.
3. Adapt existing strategies in the group to generate bacterial toxoids covalently attached to Adhirons
4. Demonstrate ER-mediated delivery of the complex and specific labelling of relevant subcompartments

Novelty The project will both exemplify the state-of-the-art in protein labelling technology and illustrate the power of the Leeds-derived Adhiron technology in cellular labelling. The project will enable a potential step-change in cellular imaging technology by enabling reproducible, clean labelling of cellular compartments in live cell imaging approaches without the need for genetic modification.

Timeliness The project builds on a number of recently developed technologies in the group of MEW, WBT and MM and will lead to exemplification of a new technology for cellular labeling with wide potential application in studies of vesicle trafficking, viral infection and disease biology. The approach can be rapidly adapted to suit super-resolution approaches (by choice of suitable dyes).

Experimental approach The approach taken is summarized in the objectives section and will take advantage of recent experience in protein labelling using sortase and/or Amber suppression, expression and purification of modified toxins and experience with imaging approaches to the study of toxin trafficking.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 01/10/2015 30/09/2023
1775197 Studentship BB/M011151/1 01/10/2016 31/03/2021 Tomasz Kaminski