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 The first stage of this project has involved two important milestones. Firstly, working closely with imaging experts of the advanced imaging center at HHMI Janelia, we have thoroughly designed our high end imaging applications. The imaging facility at Janelia is the best in the world, containing microscopes capable of imaging faster, deeper and or at higher resolution than is commercially available. The experimental design is therefore key to harnessing the power of these facilities. Working in person and by telephone with the AIC director, we have designed a new set of constructs that we are now testing ahead of starting imaging later in this project. The design of these has also involved imaging scientists at our own institution who have benefited from working with a world leader. Imaging with these new tools has generated some data but progress has been slowed due to COVID shutting down of laboratory spaces. Moreover we have been unable to travel to Janelia due to travel restrictions.

The second milestone involved local training workshop for imaging scientists in the UK. This was highly successful, sold out 3 day course run by the director AIC. It involved faculty, staff and PhD students from a number of disciplines (biology, physics, engineering and chemistry) from 3 HE institutions as well as industry. During this time, the AIC director also met one on one with most delegates to help plan their imaging experiments. The knowledge transfer was extremely large. We also recorded aspects of the training and acquired the teaching materials so that local imaging experts can use them to run the course in the future. Feedback was overwhelmingly positive and we aim to re-run this course or a variant of it later in the course of the grant.

Objective three involved generation CRISPR modified cell-lines for use in superresolution imaging. We succesfully made a LAMB1-Dendra2 line and verified its expression,. However, very dissapointly, the tagged protein was not efficiently secreted meaning it could not be used for our studies. We published these data as negatve findings in BMC Genomics as the rationale for the design was likely to be used for others.

This failure has not put us off. Instead, again working with Janelia, we have designed two new constructs. A lentiviral expression system to express LaNt a31 with photoactivable mCherry and a CRISPR modified LAMB3 c-terminal PA-GFP tag. The first of these was complete in July 2019 and we have tested the outcome and everything appears to work as expected. The second tool, was the focus of work up to February 2020; however, this has stop in March 2020 due to COVID and has been extremely problematic in restarting. In late 2021, early 2022 we now have validated tools ready for both N and C terminal tags on LAMB3. These constructs express in cells in a dish, are photoactivatable and, most importantly, are deposited by epidermal and corneal keratinocytes.

As these tool are now ready and validated we will apply for time at AIC to complete objectives relating to image acquisition and analysis. AIC has recently begun allowing external researchers to apply again and we are hopeful to be ready to submit for the next round.
Exploitation Route Resources from training workshop can be widely shared. Our experiments are ongoing but the constructs we are generating will be made available to other scientists
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...