Basement membranes generally determine different tissue compartments in complex organs, such as skin, playing not only an important structural but also a regulatory role. We have previously demonstrated the formation of a regular basement membrane in organotypic three-dimensional (3D)-cocultures of human skin keratinocytes and fibroblasts by indirect immunofluorescence and transmission electron microscopy. In this assembly process, cross-linking of type IV collagen and the laminin γ1 chain by nidogen is considered a crucial step. For a functional proof, we have now competitively inhibited nidogen binding to laminin in 3D-cocultures with a recombinant laminin γ1 fragment (γ1III3-5 module) spanning this binding site. Repeated treatment abolished the deposition of nidogen at the epithelial-matrix interface but also greatly perturbed the presence of other matrix constituents such as laminin and perlecan. This effect persisted over the entire observation period of 10 to 21 days. In contrast, some components of the basement membrane zone were only moderately affected, with the laminin-5 isoform (γ2 chain), type IV collagen and integrin α6ß4 still showing a distinct staining at their regular position, when seen by light microscopy. Furthermore, epidermal morphology and differentiation remained largely normal as indicated by the regular location of keratins K1/K10 and also of late differentiation markers. Ultrastructural examination demonstrated that the γ1 fragment completely suppressed any formation of basement membrane structures (lamina densa) and also of hemidesmosomal adhesion complexes. As a consequence of hemidesmosome deficiency, keratin filament bundles were not attached to the ventral basal cell aspect. These findings were further substantiated by immuno-electron microscopy, revealing either loss or drastic reduction and dislocation of basement membrane and hemidesmosomal components. Taken together, in this simplified human skin model (representing a `closed system') a functional link has been demonstrated between compound structures of the extra- and intracellular space at the junctional zone providing a basis to interfere at distinct points and in a controlled fashion.

Nidogens are considered as classical linkers joining laminin and collagen IV networks in basement membranes (BMs); however, recent genetic approaches have suggested that nidogens function in a tissue-specific and developmental context. Thus, in mice lacking both nidogen-1 and -2 heart and lung were severely affected, causing neonatal death. Furthermore, in various locations, extravasation of erythrocytes was observed implying microvascular defects. Mice expressing solely either isoform, had a functional BM, although nidogen-2 binds with lower affinity to the laminin gamma1 chain. Having previously blocked BM formation by interfering with nidogen-1 binding to laminin in skin-organotypic cocultures, here we investigated the roles of nidogen-1 and -2 in this model. For that purpose, human HaCaT cells were grown in three-dimensional cocultures on collagen matrices containing murine fibroblasts of varying nidogen deficiency. As with our experiments blocking laminin-nidogen interaction, lack of both nidogens completely prevented BM deposition and ultrastructural assembly of BM and hemidesmosomes, although other BM proteins remained detectable at comparable levels with no signs of degradation. Supplementation by recombinant nidogen-1 or -2 restored these structures, as shown by immunofluorescence and electron microscopy, confirming that in this system nidogen-2 is equivalent to nidogen-1, and both can promote the development of a functional BM zone.

This article, written from the stance of a public planner and a policy maker, explores the challenges and potential in creating future learning environments through the concept of a new learning landscape. It is based on the belief that physical planning can support the strategic goals of universities. In Denmark, a political focus on education as a mean to improve national capacity for innovation and growth are redefining the universities role in society. This is in turn changing the circumstances for the physical planning. Drawing on examples of physical initiatives in three different scales--city, building and room scale, the paper highlights how space and place matters on an interpersonal, an interprofessional and a political level. The article suggests that a wider understanding of how new learning landscapes are created--both as a material reality and a political discourse--can help frame an emerging community of practice. This involves university leaders, faculty and students, architects, designers and urban planners, citizens and policy makers with the common goal of creating future learning environments today.

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