What do membranes really look like? New approaches to 3D multiplexed imaging of the cell surface
Lead Research Organisation:
University of Strathclyde
Department Name: Physics
Abstract
Proteins localised to the surface membranes of cells play key roles in normal and diseased cell biology and are the targets for the vast majority of existing pharmacological treatments. To understand these proteins on the molecular level, spatial mapping of their distribution on the surface of cells at nanometre resolution and in three dimensions (3D) is urgently required. We propose to use a combination of novel super-resolution microscopy and fluorescent labels to study the arrangement of transport proteins on the surface of fat cells. This work will thus act as a paradigm for a novel imaging approach of wide applicability and interest to academics and Big Pharma alike. Our proposal links the strategic EPSRC themes of Biophysics and Chemical Biology and will develop enabling platform technologies that will underpin many facets of life and medical sciences.
Organisations
Publications
Geiser A
(2023)
GLUT4 dispersal at the plasma membrane of adipocytes: a super-resolved journey
in Bioscience Reports
Herdly L
(2022)
Benchmarking thiolate driven photoswitching of cyanine dyes
Herdly L
(2023)
Benchmarking Thiolate-Driven Photoswitching of Cyanine Dyes
in The Journal of Physical Chemistry B
Koester AM
(2022)
GLUT4 translocation and dispersal operate in multiple cell types and are negatively correlated with cell size in adipocytes.
in Scientific reports
| Description | An unanticipated by product of this work was the development of new approaches to photoswitching dyes which will be of wide interest across the community. |
| Exploitation Route | Our studies underpin the versatile role of thiols in singlemolecule photoswitching, provide a guideline for optimizing SMLM imaging, and support the development of novel imaging buffers and dyes. |
| Sectors | Chemicals Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
| Description | How does EFR3 control insulin-stimulated plasma membrane dispersal of GLUT4? |
| Amount | £481,833 (GBP) |
| Funding ID | BB/X005178/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 04/2023 |
| End | 05/2026 |
| Description | SPRINT: A SuPer-Resolution time-resolved ImagiNg and specTroscopy facility for rapid biomolecular analysis |
| Amount | £437,498 (GBP) |
| Funding ID | BB/V019643/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 07/2021 |
| End | 05/2023 |
| Title | 3D printing new infusion chambers for super-resolution microscopes. |
| Description | Developed and refined a fully 3D printed perfusion system and chambers for <£200 which can be applied to a variety of different bioimaging experiments, dSTORM, DNA PAINT, Ca2+ imaging etc. and is compatiable with a variety of different imaging systems. By using very basic coding the perfusion system was fully automated and allowed for longterm imaging experiments to be run unattended. The perfusion system had a precision and repeatablility which was found to be comparable to 1000 uL pippettes. This system allowed DNA paint imaging to be be carried out in the labs with the ability to wash on and off different imaging strands and change the concentration of imaging strands present in the imaging chambers meaning that optimum imaging conditions and enhanced localisation precision could be obtained. |
| Type Of Material | Improvements to research infrastructure |
| Year Produced | 2023 |
| Provided To Others? | No |
| Impact | This will be elaborated in a paper presently being prepared. |
| Title | Benchmarking Thiolate-Driven Photo-switching of Cyanine Dyes |
| Description | Carbocyanines are among the best performing dyes in single-molecule localization microscopy (SMLM), but their performance critically relies on optimized photo-switching buffers. Our work provides a general strategy for achieving optimal resolution in SMLM with relevance for the development of novel buffers and dyes. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| Impact | Carbocyanines are among the best performing dyes in single-molecule localization microscopy (SMLM), but their performance critically relies on optimized photoswitching buffers. Here, we study the versatile role of thiols in cyanine photoswitching at varying intensities generated in a single acquisition by a microelectromechanical systems (MEMS) mirror placed in the excitation path. The key metrics we have analyzed as a function of the thiolate concentration are photon budget, on-state and off-state lifetimes and the corresponding impact on image resolution. We show that thiolate acts as a concentration bandpass filter for the maximum achievable resolution and determine a minimum of ~1 mM is necessary to facilitate SMLM measurements. We also identify a concentration bandwidth of 1-16 mM in which the photoswitching performance can be balanced between high molecular brightness and high off-time to on-time ratios. Furthermore, we monitor the performance of the popular oxygen scavenger system based on glucose and glucose oxidase over time and show simple measures to avoid acidification during prolonged measurements. Finally, the impact of buffer settings is quantitatively tested on the distribution of the glucose transporter protein 4 within the plasma membrane of adipocytes. Our work provides a general strategy for achieving optimal resolution in SMLM with relevance for the development of novel buffers and dyes. |
| Title | Further enhancement of DNA paint. |
| Description | Our new perfusion system allowed for dSTORM imaging buffer to be optimised during image acquisition on the fly for increased localisation precision through careful control over the buffer pH and thiolate concentration. Several different novel imaging and docking strands were also developed and needed imaging and labelling conditions optimised to increase localisation precision on single molecule surfaces down to ~ 3nm. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2024 |
| Provided To Others? | No |
| Impact | New approaches will be published for the field in 2024. |
| Title | Analysis of GLUT4 dispersal in cultured adipocytes |
| Description | Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type IIIalpha to the cell surface. Using direct stochastic reconstruction microscopy, dSTORM, we show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. Enclosed here are the dSTORM datasets and associated analyses for all experiments, presented as a single folder for each experiment. Please also refer to 'dSTORM imaging files for EFR3.. ' record for additional files and analyses. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| Impact | See publications associated with this award. |
| URL | https://pureportal.strath.ac.uk/en/datasets/3c4691b7-2f26-42fb-8886-20a7dbbe1d3f |
| Title | Data for: "GLUT4 translocation and dispersal operate in multiple cell types and are negatively correlated with cell size in adipocytes." |
| Description | Dataset for all results in Koester et al presented as a series of Prism files. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2023 |
| Provided To Others? | Yes |
| Impact | See publications listed. |
| URL | https://pureportal.strath.ac.uk/en/datasets/8a14e947-b1f1-46ed-8207-65d91259a4d4 |
| Description | Presentation at Microscopy meeting in Germany. |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Work presented in relation to single molecule imaging at an event organised by PicoQuant in Germany. |
| Year(s) Of Engagement Activity | 2022 |