Super-resolution imaging of dual photoactivatable-tagged laminin allows N- and C-terminals to be spatially resolved

"Introduction Laminins (LM) are core proteins of basement membranes (BM), providing structural support and involved in cell differentiation, migration, proliferation and stem cell maintenance. 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. Interestingly, despite missing the LN domains responsible for network self-assembly, LMα3β3γ2 is still capable of forming functional BM. One hypothesis to explain this conundrum is that LMα3β3γ2 orientation changes upon secretion from parallel to the plasma membrane to vertical and aggregated. A second explanation involves a laminin and netrin-related protein, LaNt α31, which contains an LN domain and which could allow for network assembly. Advancements in super-resolution imaging now allow for the creation of constructs to image LM deposition and dynamics with spatial 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, lung carcinoma epithelial cells and breast adenocarcinoma cells. 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. 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, deposition and co-localisation studies with LaNt α31. Furthermore, changes to the ECM due to LMβ3 and LaNt α31 proteins can now be examined using atomic force microscopy and traction force microscopy with these new exciting imaging tools."