Utilising advanced imaging techniques and super-resolution microscopy to understand basement membrane laminin network formation and remodelling

Session

Poster Session

Authors

Natasha Chavda (2), Hannah Levis (2), Colin Jahoda (1), Kevin Hamill (2)

Affiliations

1. Durham University
2. University of Liverpool

Keywords

Basement membrane, Extracellular matrix, Laminin, LaNt, Network Formation, Photoactivation, SIM, Super-resolution, TIRF

Abstract text

Introduction

The laminins (LMs) are a family of extracellular matrix (ECM) proteins and form one of the two key networks of the basement membrane (BM) structure, along with collagen IV. LMs are critically involved in maintaining tissue integrity and provide substrates for cell migration during development, cancer metastasis and wound repair. Each LM is a heterotrimer comprised of an α, a β and a γ chain (Fig. 1A). The archetypal assembled heterotrimer consists of three so-called “short arms” of 30-40nm connected to a “long arm” of ~77nm, which is followed by 5 globular domains (LG domains). LMs interact with a variety of different ECM proteins as well as cell surface receptors. The highest affinity receptor binding sites are located within the LG domain, however, further binding sites have been identified within the Laminin N terminal domains (LN) at the amino terminus of the short arms. The purpose of these N-terminal interactions are not known. Once deposited into the ECM, LMs assemble into pseudo-polymers via LN-LN interactions (Fig. 1B), via a two-step process of nucleation requiring interaction with cell-surface receptors, followed by propagation. This model has been established primarily through recombinant protein addition to cells or in non-cellular conditions, and the actual processes involved in matrix assembly have not been studied directly, primarily due to imaging limitations. Specifically, how the transition from receptor-binding but non-polymerised to network forming is not known.

LM network composition and remodelling contributes greatly to BM biophysical properties and functions. Many serious genetic disorders are associated with LM mutations, particularly those that affect polymerisation. The LMα3β3γ2 (LM332) isoform is found in epithelial BMs, including the cornea and skin, with mutations causing diseases, such as junctional epidermolysis bullosa. Interestingly, despite missing the LN domains responsible for network self-assembly, LM332 is still capable of forming functional BMs (Fig. 1C). One hypothesis to explain this conundrum is that LM332 orientation changes upon secretion from parallel to the plasma membrane, allowing the usage of the LN domain cell-surface binding sites, then to vertical and aggregated (Fig. 2). A second explanation involves a LM and netrin-related protein, LaNt α31, which contains an α-chain LN domain and which could allow for stable network assembly by providing the "missing" LN domain. Advancements in super-resolution imaging now allow for the creation of constructs to image LM deposition and dynamics with spatial z-resolutions capable for these questions to be addressed. 

Materials and Methods

Multiple photoactivatable (PA) -fluorescent expression constructs were generated for LaNt α31 and LMβ3, including with tags on both ends of the LMβ3. These were validated, via transfection into human cell lines, by western blotting and immunostaining against LMα3 and LMγ2 chains. The constructs were imaged live in super-resolution using spinning disk, lattice-SIM and TIRF microscopy. Plasmids were then packaged into lentivirus particles and stable expression lines generated in corneal epithelial cells, allowing biomechanical changes to be examined using AFM. Corneal epithelial cell lines with a HaloTagged LaNt α31 construct, along with mutant LN domain versions, have also been imaged.

Results

Western blotting confirmed expression of the predicted sized proteins. Transfected corneal epithelial cells showed deposition of PA-mCherry-LAMB3-GFP outside the cells in the archetypal LM deposition pattern and co-localisation with LMα3 and LMγ2, indicative of LM332 heterotrimer formation. Excitingly, photoactivation of PA-mCherry and PA-GFP tags identified spatial resolution between LMβ3 N- and C-terminals when imaged using the Elyra Lattice-SIM (Fig. 3). Corneal epithelial cells expressing LaNt-PAmCh cells also were successfully photoactivated (Fig. 4) and along with HaloTag-LaNt cells were both successfully imaged using Lattice-SIM, with the HaloTag cells in particular expressing a strong fluorescent signal (Fig. 5). Early AFM data suggests a difference in stiffness between wildtype and LaNt-PAmCh corneal epithelial cells.

Discussion

Confirmation that the dual tags can be spatially resolved and the generation of stable expression cell lines will allow further investigation, using super-resolution techniques, of LM332 secretion and deposition by allowing LM orientation changes to be observed.  Co-localisation studies with LaNt α31, along with other key ECM proteins, can also be carried out using the PAmCh and HaloTag proteins. Furthermore, changes to the ECM due to LMβ3 and LaNt α31 proteins has been shown and will be further examined using AFM and TFM.

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