Fluorescently tagged laminin subunits facilitate analyses of the properties, assembly and processing of laminins in live and fixed lung epithelial cells and keratinocytes

Abstract

Recent analyses of collagen, elastin and fibronectin matrix assembly, organization and remodeling have been facilitated by the use of tagged proteins that can be visualized without the need for antibody labeling. Here, we report the generation of C-terminal tagged, full-length and "processed" (α3ΔLG4-5) human α3 as well as C-terminal tagged, full-length human β3 laminin subunits in adenoviral vectors. Human epidermal keratinocytes (HEKs) and human bronchial epithelial (BEP2D) cells, which assemble laminin-332-rich matrices, as well as primary rat lung alveolar type II (ATII) cells, which elaborate a fibrous network rich in laminin-311, were infected with adenovirus encoding the tagged human laminin subunits. In HEKs and BEP2D cells, tagged, full-length α3, α3ΔLG4-5 and β3 laminin subunits incorporate into arrays of matrix organized into patterns that are comparable to those observed when such cells are stained using laminin-332 subunit antibody probes. Moreover, HEKs and BEP2Ds move over these tagged, laminin-332-rich matrix arrays. We have also used the tagged β3 laminin subunit-containing matrices to demonstrate that assembled laminin-332 arrays influence laminin matrix secretion and/or assembly. In the case of rat ATII cells, although tagged α3 laminin subunits are not detected in the matrix of rat ATII cells infected with virus encoding full-length human α3 laminin protein, processed human α3 laminin subunits are incorporated into an extracellular fibrous array. We discuss how these novel laminin reagents can be used to study the organization, processing and assembly of laminin matrices and how they provide new insights into the potential functional importance of laminin fragments.

Focal Contact and Hemidesmosomal Proteins in Keratinocyte Migration and Wound Repair

“…6 Moreover, live cell imaging of keratinocytes expressing a tagged subunit of the laminin-332 protein demonstrates that migrating cells deposit laminin-332 in linear arrays or tracks along which they move. 106,145 Laminin-332 containing an unprocessed c 2 subunit has also been reported to drive the hypermotility of keratinocytes. 146 Moreover, there is evidence that matrices rich in laminin-332 containing uncleaved a 3 subunits support migration and that they do so in an a 3 b 1 integrin-dependent manner.…”

Laminin deposition in the extracellular matrix: a complex picture emerges

“…This involves visualizing the deposition of fluorescently tagged laminin subunits onto the substrate of live cells (Fig. 3A,B) (Hopkinson et al, 2008;Sehgal et al, 2006). Using these tagged molecules, one can observe the deposition of nascent laminin subunits together with their receptor molecules in real time.…”

“…Indeed, this approach to the study of laminin-matrix formation is already providing new insights. For example, we have used live-cell imaging of tagged laminin subunits to demonstrate that truncated versions of the a3 laminin subunit are incorporated into the matrix of epithelial cells, at least in cells that also express wild-type laminin-332 subunits (Hopkinson et al, 2008). In addition, we have shown that matrix deposition is regulated by a feedback loop when cells are plated on, or move onto, an already assembled lawn of matrix proteins (Hopkinson et al, 2008).…”

“…For example, we have used live-cell imaging of tagged laminin subunits to demonstrate that truncated versions of the a3 laminin subunit are incorporated into the matrix of epithelial cells, at least in cells that also express wild-type laminin-332 subunits (Hopkinson et al, 2008). In addition, we have shown that matrix deposition is regulated by a feedback loop when cells are plated on, or move onto, an already assembled lawn of matrix proteins (Hopkinson et al, 2008). Imaging laminin-matrix formation in live cells is a perfect complement to the many biochemical and molecular approaches to the study of matrix formation and protein interactions that have been applied over the last 20 years.…”

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

“…Recently, a series of elegant studies in C. elegans described genetically tagging the C-terminus of the worm LMβ chain ortholog with fluorescent proteins, allowing in vivo observation of BM turnover and remodelling [13,14]. In human cells, adenoviral-mediated expression of the two smallest LMs, LMβ3 and LMγ2, with C-terminal fluorescent tags has also been performed [15,16]. However, these constructs are at the adenoviral packaging limit and allow only transient expression.…”