Super-resolution imaging of laminin deposition and organisation

Lead Research Organisation: University of Liverpool
Department Name: Institute of Ageing and Chronic Disease

Abstract

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Description This project was designed to use recent advances in live cell imaging to answer questions about how basemente membranes form in terms of laminin organisation and interaction with LaNts. To do this we collaborated with the advanced imaging centre (AIC) at Janelia, a Howard Hughes Medical Institute funded facility that is the world leader in light microscopy. As part of the grant, the director of the AIC ran a week-long training workshop for microscopists at the University of Liverpool and also rann a series of design and planning sessions with local researchers. Following these planning meetings, we designed, constructed and validated a series of constructs to allow us to ask our biological questions. This proved challenging with lots of iterative improvements in design required due to issues in secretion and tag interaction that were not anticipated. This grant straddled the COVID lockdown periods which sorely hampered progress, both from a technical perspective but also in terms of personnel. Specifically, the people working on this project's contracts finished during the disrupted year and (as this grant did not cover salary) the people who joined the project later required more training and development. During COVID and the years following, the AIC did not accept research groups and only really fully opened in the last 12 months.

Despite these challenges, we have now a suite of validated tools for superresolution imaing of LaNts and laminins. We have submitted to AIC to use their facilities in 2024. This is after the end date of the grant but it is very pleasing to be able to move forward.
Exploitation Route The tools we have made will be used by laminin researchers around the world.
Sectors Education

Healthcare

Pharmaceuticals and Medical Biotechnology
 
Title CRISPR constructs - LAMA5 and LAMB1 mutations 
Description CRISPR guide and donor plasmids to genetically engineer cell lines to carry mutations in laminin alpha5 or laminin b1 encoding genes. These mutations correspond to icritical residues within these proteins that are required for laminin to laminin interaction and therefore network assembly. The mutations are equivalent to those described in other laminins in human genetic diseases merosin deficient congenital muscular atropy and Pierson syndrome respectively. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact Ongoing. Generation of cell line that makes not network forming laminins allows dynamic analysis of laminin BM assembly, dissection of role of network assembly status in growth factor sequestration, outside-in and inside-out signaling and traction force generation. 
 
Title CRISPR tools for Dendra2 tagged laminin generation 
Description CRISPR guide RNA and donor plasmid to genetically modify cell lines to harbour a fluorescent tag on C-terminus of laminin beta1 gene. Fluorescent tag is photoconvertible Dendra2. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact Ability to image in live cells native laminin deposition, turnover and dynamics. Suitable for use in super-resolution experiments. Allows long-term, dynamic analysis of basement membrane assembly. Also potential for use in 3D systems eg for tumour invasion and metastasis assays. Required reagent to progress with imaging experiments 
 
Title LAMB3-PA-GFP cell line 
Description CRISPR modified cell line (hTCEpi - human corneal epithelial cells) engineered to express laminin beta3 with a C-terminal photoactivatable GFP tag 
Type Of Material Cell line 
Year Produced 2020 
Provided To Others? No  
Impact New line. Impact will follow including publication. 
 
Title Lentiviral construct - LaNt a31 PA mcherry 
Description lentiviral particles to deliver human lama3ln1 (encoding LaNt a31) with C-terminal photoactivatable mCHerry protein. Allows stable expression of this tagged protein in mammalian cells. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact Will allow superresolution imaging of LaNt a31 
 
Title Plasmid- LaNt a31 dendra2 
Description plasmid encoding LaNt a31 protein tagged at C terminus with dendra2 photoconvertible fluorescent tag. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact None yet. Required for planned super-resolution experiments 
 
Description HHMI Janelia Imaging Centre 
Organisation Howard Hughes Medical Institute
Department Janelia Research Campus
Country United States 
Sector Academic/University 
PI Contribution Hosting and on-site organisation of a 3 day training workshop. Access to computer suite, lecture theatres etc
Collaborator Contribution On-site 3 day training workshop for 30 delegates at University of Liverpool run by director of AIC-Janelia facility Dr Teng Leong Tew. Attendees included University of Liverpool, Manchester and LJMU staff and PhD students as well as industry professionals from Lever. In addition, Leong engaged in a number of one on one experimental design consultancy sessions directly aiding not only my team but also other researchers in the design of their super-resolution and light sheet microscopy experiments.
Impact Improved design and analysis of imaging-based experimentation. Design of super-resolution and other high end imaging modality experiments. High level training of next generation of imaging scientists. Applications in preparation to use state of the art facilities at AIC Janelia.
Start Year 2017
 
Description Raphael Reuten - LaNt protein purification 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution LaNt expressing plasmids
Collaborator Contribution Purified Lant a31 proteins
Impact Purified protein - required for planned experiments involving exogenous addition of protein for imaging experiments
Start Year 2017
 
Description Museum events - British Science Week, Meet the Scientists 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Multiple events
Ran interactive stands designed to stimulate engagement with science. Included a giant cardiovascular model made from air conditioning ducting, UV-sensitive camera imaging of participants as well as microscopy and slide viewing
Year(s) Of Engagement Activity 2018
URL https://lantsandlaminins.com/2018/06/23/inspiration-and-fun-at-the-world-museum/
 
Description School Visit - Live cell imaging demonstrations 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Multiple visits to our research institute of groups of primary school age children and, separately, high school age children. Live demonstrations of microscopy in practice; combination of static histology slide imaging and live imaging of fluorescent protein expressing cells. Accompanied by other activities; eye dissection, cardiovascular modelling and core biology teaching.
Year(s) Of Engagement Activity 2017,2018
URL https://lantsandlaminins.com/2018/02/08/inspiring-the-next-generation-of-scientists-with-hands-on-ac...