Biological physics of protein clustering in epigenetic memory and transcriptional control
Lead Research Organisation:
University of York
Department Name: Physics
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
Organisations
People |
ORCID iD |
| Mark Leake (Principal Investigator) |
Publications
Badrinarayanan A
(2022)
Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex in Live Escherichia coli Bacteria.
in Methods in molecular biology (Clifton, N.J.)
Dilliway C
(2022)
Working at the interface of physics and biology: An early career researcher perspective.
in iScience
Dresser L
(2021)
Amyloid-ß oligomerization monitored by single-molecule stepwise photobleaching.
in Methods (San Diego, Calif.)
Dresser L
(2022)
Tween-20 Induces the Structural Remodeling of Single Lipid Vesicles.
in The journal of physical chemistry letters
Evans D
(2023)
GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms
in Microbial Cell
Hunter P
(2022)
Single-molecule and super-resolved imaging deciphers membrane behavior of onco-immunogenic CCR5.
in iScience
Jin X
(2021)
Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness.
in Science advances
Laidlaw KME
(2021)
A glucose-starvation response governs endocytic trafficking and eisosomal retention of surface cargoes in budding yeast.
in Journal of cell science
| Title | Tools to quantify molecular tracks in vivo within Liquid-Liquid Phase-Separated condensates |
| Description | Poster presented as part of the Crick BioImage Analysis Symposium. In soft matter biophysics, LLPS is a surprising effect in which a solution spontaneously demixes into droplets. In biology, there is mounting evidence that LLPS underpins both normal physiology (e.g., immune responses) and disease (e.g. cancer and neurodegeneration). I present two examples of LLPS from different biological kingdoms: pathogenic bacteria demonstrating antibiotic resistance and plants regulating their epigenetic response to winter cold in order to flower. However, due to the complex out-of-equilibrium dynamics involved, the underlying physical principles of biological LLPS are largely unknown. To gain a better understanding, we need specialised microscopy and post-processing tools that can observe and describe LLPS over a broad range of lengths and timescales. I introduce an overview of our bespoke and open-source software tools to process specialized LLPS microscopy images: i) single-molecule tracking and ii) simulated collective fluorescent bleaching and recovery to extract the physics behind the biological phenomenon of membraneless droplets inside cells. Permission has been given by authors to upload to Crick Figshare. Copyright remains with the original holders. |
| Type Of Art | Film/Video/Animation |
| Year Produced | 2023 |
| URL | https://crick.figshare.com/articles/poster/Tools_to_quantify_molecular_tracks_in_vivo_within_Liquid-... |
| Description | We developed a new type of light microscopy to allow us to track key proteins in root tips that are involved in regulating whether or not plants flower (a process called vernalization). We have found clear evidence that two of these proteins called Vin3 and Vrn5 operate in clusters of typically 5-20 molecules. This ties in really well with theory modeling that suggested that a "memory" effect for this type of flowering regulation must be due to their being multiple copies of the memory elements. These findings may tell us how general gene memory modifications (so called "epigenetics") are regulated in multiple different organisms, including increasing our understanding of the onset of human diseases such as cancer. |
| Exploitation Route | This work will offer new insights into researchers studying how genes are regulated in living cell. It will also inform researchers who wish to understand how genetic disease start, and how they might be prevented. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| URL | https://sites.google.com/a/york.ac.uk/mark-leake-group/home |
| Description | Findings concerning biomolecular condensate formations in root tips in this project helped in part stimulate preliminary conversation with a non-academic partner FujiFillm Diosynth Biotechnologies (https://fujifilmdiosynth.com/), which ultimately led to their becoming a project partner on a new EPSRC Open Fellowship which I have beened awarded due to formally start ca. April 2024 |
| First Year Of Impact | 2023 |
| Sector | Healthcare |
| Title | Datasets for "Single-molecule and super-resolved imaging deciphers membrane behaviour of onco-immunogenic CCR5" |
| Description | Flow cytometry Modality / instrument: Flow cytometer (CytoFLEX LX, Beckman Coulter) File format: FCS + XIT (CytExpert, Beckman Coulter). Samples and acquisitions: Fluorescent fusions in live Chinese Hamster ovary (CHO) cells. File Cell line Runs Cells counted CONTROL.fcs CHO wild-type 1 7000 GFP-CCR5.fcs CHO-GFP-CCR5 1 7000 Exp_20220916_1_GFP.xit N/A - metadata Approx. size 6 MB PaTCH microscopy images Imaging modality / instrument: Brightfield + PaTCH fluorescence microscopy Image format: OME TIFF (16 bit) + MicroManager metadata files Microscope settings: 488 nm triggered excitation; split red/green detection, cropped to green (GFP) channel only; 10 ms/frame laser exposure; 13.5 ms/frame-to-frame; 53 nm/px. Photometrics Prime95b CMOS. Samples and acquisitions: Fluorescent fusions of GFP-CCR5 receptor in live CHO cells imaged with and without 100 nM CCL5 ligand. Each subfolder corresponds to a field of view and contains one brightfield and one PaTCH acquisition of the same cell. Folder Condition Fields of view AC6 CONTROL sc CCL5- 11 AC6 CCL5 sc CCL5+ (100 nM) 10 Approx. size before compression: 14 GB Structured illumination microscopy - volumetric stacks Imaging modality / instrument: SIM fluorescence microscopy (custom setup at NPL based on Olympus IX71) Image format: OME TIFF (16 bit) with intrinsic metadata (voxel size) Microscope settings: 638 nm excitation; 60x/1.3 NA; Flash 4.0, Hamamatsu Photonics. For additional details see the reference below (Hunter et al, bioRxiv). Samples and acquisitions: Dylight 650-MC-5 labeled CCR5 receptor in fixed CHO-CCR5 cells, imaged with and without 100 nM CCL5 ligand. Each acquisition is of a unique field of view and contains one SIM reconstruction as an XYZ volumetric stack. 'Basal membrane' acquisitions consist of 5 slices at 200 nm z-intervals across the range of the basal membrane. 'Whole cell' acquisitions are made up of 7 slices with 500 nm z-interval ranging from just below the basal membrane to just above the apical membrane. Folder Subfolder/condition Fields of view Basal membrane CCL5- 5 Basal membrane CCL5+ (100 nM) 6 Whole cells CCL5- 5 Whole cells CCL5+ (100 nM) 8 Approx. size before compression: 300 MB |
| Type Of Material | Database/Collection of data |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| URL | https://zenodo.org/record/7082977 |
| Title | Slimfield: Escherichia coli DNA repair proteins (RecA-mGFP and RecB-sfGFP) |
| Description | Imaging modality / instrument: Brightfield + Slimfield Image format: OME TIFF (16 bit) + MicroManager metadata files Microscope settings: 488 nm triggered excitation; split detection, cropped to GFP or RFP/GFP (left/right) channels; 3 ms/frame laser exposure; Photometrics Prime95b CMOS Samples and acquisitions: Fluorescent fusions in live E.coli cells. MMC = mitomycin C No. fields of view MMC- MMC+ (0.5 ug/ml 3h) RecA-mGFP 7 15 RecB-sfGFP 17 21 MG1655 control 10 10 Approx. size before/after compression: 21 GB / 7 GB |
| Type Of Material | Database/Collection of data |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| URL | https://zenodo.org/record/6639100 |