CRISPR-Cas9-mediated labelling of the C-terminus of human laminin ß1 leads to secretion inhibition.

OBJECTIVES: The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. Recently, the nematode C. elegans ortholog of human the LMß1 chain was labelled at the C-terminus with the photoconvertible fluorophore Dendra2. Here we used genome editing to establish a similar system in a mammalian cell line as proof of concept for future mammalian models. RESULTS: CRISPR-Cas9 was used to introduce the Dendra2 sequence at the C-terminus of LMß1 in the human lung adenocarcinoma cell line A549. Despite expression of the tagged protein within cells, no detectable LMß1-Dendra2 protein was deposited to the extracellular matrices or conditioned media of edited cells. Moreover, the edited cells displayed reduced proliferation rates. Together, these data suggest that, in humans, addition of C-terminal Dendra2 tag to LMß1 inhibits LM secretion, and is not a viable approach for use in animal models.

The alpha-3 polypeptide chain of laminin 5: insight into wound healing responses from the study of genodermatoses.

Laminin 5 (kalinin/epiligrin/nicein) is an essential structural component of the dermal-epidermal junction, composed of three polypeptide subunits: laminin alpha3, beta3 and gamma2. Studies of the inherited skin fragility disorder junctional epidermolysis bullosa (JEB) have suggested that the major role of this heterotrimeric protein is to act as an adhesive ligand essential for binding the epidermis to the underlying dermis and thus maintaining the integrity of the skin. Protein interaction studies have shown that the C terminus of the alpha3 subunit binds to a range of integrin complexes depending on the motility status of keratinocytes. This allows laminin 5 to interact with either hemidesmosomes or the actin cytoskeleton. Recently we have reported that the absence of the N-terminal region of laminin alpha3a in laryngo-onchyo-cutaneous syndrome causes excessive granulation tissue production at wound sites. As granulation tissue production is also a problem in JEB, this implicates laminin 5 in control of this wound healing response.