CRISPR-Cas9-mediated labelling of the C-terminus of human laminin β1 leads to secretion inhibition

Abstract

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 LMβ1 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 LMβ1 in the human lung adenocarcinoma cell line A549. Despite expression of the tagged protein within cells, no detectable LMβ1-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 LMβ1 inhibits LM secretion, and is not a viable approach for use in animal models.

Visualization of basement membranes by a nidogen-based fluorescent reporter in mice

“…Although genetic tagging of BM-specific proteins has been achieved in Drosophila and Caenorhabditis elegans [7] , [11] , [12] , [13] , genetic modification of BM-specific proteins in mammalian cells often disturbs their expression, function, or both [29] , [30] . Shaw et al reported that genetic tagging of the laminin β1 subunit on its C-terminus with a photoconvertible fluorescent protein prevented the subunit from being secreted from the human lung carcinoma cell line A549, even though it was translated in the cells [31] . We also attempted to tag the laminin α5 subunit with EGFP at the N-terminus.…”

A Window into Mammalian Basement Membrane Development: Insights from themTurq2-Col4a1Mouse Model

“…However, until recently, the key bottleneck in our understanding of mammalian, and indeed vertebrate BMs, has been the lack of models in which endogenous BM components are labelled. Several attempts to generate such models have been unsuccessful (Shaw et al, 2020), and in vivo studies have relied upon knock-in reporters which do not necessarily reflect endogenous protein expression and can be expressed ectopically and/or interfere with native protein function (Yamaguchi et al, 2022, Sztal et al, 2011, Futaki et al, 2023). The recent success by Morgner et al (2023) in generating a Lamb1-Dendra2 mouse model provided proof-of-principle that fluorescently tagging BM components in mouse is feasible, and in this study, we have advanced one step further by generating a mouse line in which the BM ubiquitous COL4A1 is labelled.…”

“…One possible explanation for the absence of homozygous animals is that heterotrimers containing two mTurq2-COL4A1 molecules (collagen α1/α1/α2 (IV)) may cause problems with secretion; mutant COL4A1 or COL4A2 has been shown to accumulate within cells, reducing secretion and leading to extracellular deficiency (Jeanne et al, 2015). Inhibited secretion was also observed with C-terminal tagged human laminin β-1 in adenocarcinoma cells (Shaw et al, 2020). Alternatively, given that the 7S domain is important for crosslinking collagen IV into a lattice network, it may be that two mTurq2 molecules per collagen IV trimer interferes with 7S-mediated crosslinking, but one mTurq2 per trimer can be tolerated.…”

Col4a2-eGFPmouse model reveals the molecular and functional dynamics of basement membrane remodelling in hair follicle morphogenesis

“…In C. elegans, mNeonGreen was inserted at the N-terminus of the collagen IV α2/let-2 gene, resulting in viable heterozygotes but nonviable homozygotes 14 . There was an attempt to adapt the Dendra2 tagging of laminin b1 used in C. elegans 53 to mammalian cells, but secretion failed 19 . More recently, a knock-in mouse model that expresses endogenously tagged laminin b1-Dendra2 fusion protein has been developed and used to identify BM-producing origins and dynamics in breast tumours 21 .…”

“…These properties include 1) large modular structures, 2) specialized intracellular transport and secretory mechanisms, 3) post-translational processing, 4) the assembly of supramolecular complexes, 5) complex molecular interactions and 6) unique extracellular physicochemical environments, such as redox status. While a few individuals of C. elegans and Drosophila have been manipulated to express fluorescently tagged endogenous BM proteins with normal functions 14,18 , mice expressing fluorescent tag-fused core BM proteins have exhibited functional abnormalities [19][20][21] . Therefore, generating mice that express endogenous core BM proteins fused with a fluorescent tag while maintaining normal function remains a major challenge.…”