PT  - JOURNAL ARTICLE
AU  - Valentina Iorio
AU  - Lee D. Troughton
AU  - Valentina Barrera
AU  - Kevin J. Hamill
TI  - LaNt α31 modulates LM332 organisation during matrix deposition leading to cell-matrix adhesion and migration defects
AID  - 10.1101/617597
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 617597
4099  - http://biorxiv.org/content/early/2019/04/24/617597.short
4100  - http://biorxiv.org/content/early/2019/04/24/617597.full
AB  - The laminin N terminus (LaNt) proteins are a family of laminin and netrin related proteins derived by alternative splicing from laminin encoding genes. There are few reports to date on LaNt protein function. However, one family member, LaNt α31, has been shown to regulate epidermal keratinocyte migration and adhesion, although the mechanism for these effects is unknown. Based on its protein architecture, we predicted that LaNt α31 would influence laminin organisation. To test this prediction, we induced expression of GFP tagged LaNt α31 via adenoviral delivery into human epidermal and corneal keratinocytes. Consistent with the LaNt/laminin interaction hypothesis, LaNt α31 GFP codistributed with laminin β3 in the extracellular matrix and formed a complex together as indicated by co-immunoprecipitation, while live cell assays revealed the proteins to be deposited together during new matrix synthesis. Moreover, the induced expression of LaNt α31 led to changes to laminin α3 organisation into tight clusters compared with wide arcs in control cells. These changes were associated with early maturation of hemidesmosomes and mislocalization of focal adhesion complexes. At the cellular level, epithelial cells expressing LaNt α31 GFP displayed decreased scratch closure and single cell motility rates and increased cell spread area. These differences were rescued through the provision of a pre-formed cell-derived matrix. Together these data identify a new mechanism that influences the early stages of laminin matrix assembly and adds the LaNt proteins as new players in defining cell-to-matrix adhesion and migration characteristics.Grant support This work was supported by funding from the Biotechnology and Biological Sciences Research Council (BB/L020513/1, KH), Fight For Sight (New lecturers’ award, KH), British Skin Foundation (PhD studentship, KH) and National Institute of Arthritis Musculoskeletal and Skin (K99/R00 1K99AR060242, KH).HighlightsLaNt α31 is a new mediator of laminin-dependent processes.LaNt α31 codistributes and is deposited with laminin 332 in the extracellular matrix of epithelial cells.Increased LaNt α31 expression leads to changes in laminin 332 organisation into tight clusters compared with broad tracks.Epithelial cells expressing increased LaNt α31 exhibit early hemidesmosome maturation and mislocalization of focal adhesion complexes.LaNt α31 overexpression decreases scratch wound closure rates and single cell migration rates of epithelial keratinocytes which is rescues through the provision of a preformed matrix.HighlightsThis manuscript identifies a new way in which laminin deposition and organisation is controlled, via LaNt α31. Specifically, these data demonstrate that this relatively unstudied laminin-gene splice isoform, induces the formation of laminin 332 clusters during times of new matrix synthesis, leading to changes in cell-matrix adhesion and behaviour.LMlamininhTCEpihuman telomerase reverse transcriptase immortalised corneal epithelial cellsLNlaminin N-terminal domainLaNtlaminin N-terminus proteinHDhemidesmosomeFAfocal adhesionHaCaThuman adult low calcium high temperature epidermal keratinocytesECMextracellular matrixBMbasement membraneeGFPenhanced green fluorescent proteinIPimmunoprecipitationIBimmunoblotLElaminin-type epidermal growth factor-like domainLGlaminin globular domainTIRFtotal internal reflection microscopy