The role of Fras1/Frem proteins in the structure and function of basement membrane

Basement membranes constitute architecturally complex extracellular matrix (ECM) protein networks of great structural and regulatory importance. Recently, a novel group of basement membrane proteins, Fras1 (Fraser syndrome protein (1) and the Fras1-related extracellular matrix proteins Frem1, Frem2 and Frem3, has emerged. They comprise components of the sublamina densa region and contribute to embryonic epithelial-mesenchymal integrity. Fras1/Frem share common polypeptide repetitive motifs with possible interactive and organizing functions. Mutations in genes encoding Fras1, Frem1 and Frem2 are causative for dermal-epidermal detachment in the plane of sublamina densa and have been identified in different classes of mouse bleb mutants, the murine model of human Fraser syndrome, the hallmark phenotypic characteristics of which are embryonic skin blistering, cryptophthalmos and renal agenesis. Indeed, defects in FRAS1 and FREM2 have been identified in Fraser syndrome patients. The phenotypic similarity of mouse bleb mutant strains can be attributed to the fact that Fras1, Frem1 and Frem2 have been experimentally shown to interact, forming a mutually stabilized protein complex, while Frem3, which has not yet been associated with any of the existing known mutations, operates in a more independent fashion. Fras1/Frem have been recently proposed to compensate for the activity of collagen VII, a major anchoring component of the sublamina densa, the levels of which rise only during late embryonic life. By focusing on the aforementioned data, in this review we will summarize the current knowledge about Fraser syndrome proteins and describe their contribution to basement membrane biology.     

Spatiotemporal distribution of Fras1/Frem proteins during mouse embryonic development

The Fras1/Frem gene family encodes for structurally similar, developmentally regulated extracellular matrix proteins. Mutations in Fras1, Frem1 and Frem2 have been identified in different classes of mouse bleb mutants, while defects in the human orthologs FRAS1 and FREM2 are causative for Fraser syndrome. The hallmark phenotypic feature of bleb mice is embryonic skin blistering due to dermal-epidermal detachment. The similarity of the phenotypic characteristics among the bleb mouse mutants, together with the fact that Fras1/Frem proteins are co-localized in embryonic epithelial basement membranes, suggest that they operate in a common pathway. Here, we report for the first time the immunofluorescence pattern of Frem3 and provide a comparative analysis of the spatiotemporal localization of all Fras1/Frem proteins during mouse embryonic development. We demonstrate their overall co-localization in embryonic epithelial basement membranes, with emphasis on areas of phenotypic interest such as eyelids, limbs, kidneys, lungs and organs of the gastrointestinal tract and the central nervous system. We further studied collagen VII, impairment of which produces dystrophic epidermolysis bullosa, a postnatal skin blistering disorder. We show that basement membrane levels of collagen VII rise at late embryonic life, concomitant with descending Fras1/Frem immunolabeling.     

Basement membrane localization of Frem3 is independent of the Fras1/Frem1/Frem2 protein complex within the sublamina densa

The Fraser syndrome protein Fras1 and the structurally related proteins Frem1, Frem2 and Frem3 comprise a novel family of extracellular matrix proteins implicated in the structural adhesion of the embryonic epidermis to the underlying mesenchyme. Fras1, Frem1 and Frem2 have been shown to be simultaneously and interdependently stabilized in the basement membrane by forming a ternary complex located underneath the lamina densa. However, the functional relationships between Frem3 and the other Fras1/Frem proteins remain unknown. Here we show that in the absence of Fras1 the basement membrane localization of Frem3 remains unaffected in contrast to Frem1 and Frem2 which are completely abolished from the basement membrane. This indicates that although Frem3 is localized in the sublamina densa similar to Fras1, Frem1 and Frem2 yet it is anchored in the basement membrane independently. We further demonstrate that loss of Fras1 results in the accumulation of Frem2 within epithelial cells. This finding reveals that Fras1 is not only essential as a component of a macromolecular complex for the extracellular stabilization of Frem2 but it is also required for its proper intracellular trafficking and export from embryonic epithelial cells.     

Differential localization profile of Fras1/Frem proteins in epithelial basement membranes of newborn and adult mice

The Fras1/Frem gene family encodes for structurally similar proteins of the extracellular matrix, functionally correlated with embryonic dermal-epidermal adhesion as deduced from the appearance of sub-epidermal blisters in mouse mutants compromising the function of Fras1, Frem1 and Frem2 proteins. Mutations in the human counterparts FRAS1 and FREM2 have been detected in patients suffering from Fraser syndrome. So far, Fras1/Frem proteins have been shown to be strictly colocalized in the sublamina densa of mouse epithelial basement membranes during development. Here, we focused on the characterization of the localization pattern of the aforementioned proteins, in various parts of the adult mouse skin as well as a range of organs and tissues. Frem3 was present in a broad range of epithelial basement membranes where Fras1, Frem1 and Frem2 were missing. The localization profile of Frem3 coincided with that of collagen VII in all skin basement membranes but differed in that Frem3 was additionally found in the basement membrane of several internal epithelia, where collagen VII was absent. Fras1 and Frem2 were colocalized with Frem3 in the basement membrane of certain skin parts, underlying the thin-layer, of rapidly proliferating keratinocytes, whereas Frem1 was detected only in the basement membrane of the tail. The localization pattern of Fras1 and Frem2 was indistinguishable, while both proteins along with Frem3 could be detected even in the absence of Frem1.     

Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development

Frem1 belongs to a family of structurally related extracellular matrix proteins of which Fras1 is the founding member. Mutations in Fras1 and Frem1 have been identified in mouse models for Fraser syndrome, which display a strikingly similar embryonic skin blistering phenotype due to impaired dermal-epidermal adhesion. Here we show that Frem1 originates from both epithelial and mesenchymal cells, in contrast to Fras1 that is exclusively derived from epithelia. However, both proteins are localized in an absolutely overlapping fashion in diverse epithelial basement membranes. At the ultrastructural level, Frem1 exhibits a clustered arrangement in the sublamina densa coinciding with fibrillar structures reminiscent of anchoring fibrils. Furthermore, in addition to its extracellular deposition, around E16, Frem1 displays an intracellular distribution in distinct epidermal cell types such as the periderm layer and basal keratinocytes. Since periderm cells are known to participate in temporary epithelial fusions like embryonic eyelid closure, defective function of Frem1 in these cells could provide a molecular explanation for the "eyes open at birth" phenotype, a feature unique for Frem1 deficient mouse mutants. Finally, we demonstrate loss of Frem1 localization in the basement membrane but not in periderm cells in the skin of Fras1(-/-) embryos. Taken together, our findings indicate that besides a cooperative function with Fras1 in embryonic basement membranes, Frem1 can also act independently in processes related to epidermal differentiation.     

Why mussels stick together: spatial self-organization affects the evolution of cooperation

Cooperation with neighbours may be crucial for the persistence of populations in stressful environments. Yet, cooperation is often not evolutionarily stable, since non-cooperative individuals can reap the benefits of cooperation without having to pay the costs associated with cooperation. Here we show that active aggregation leading to self-organized spatial pattern formation can promote the evolution of cooperativeness. To this end, we study the effect of movement strategies on the evolution of cooperation in mussel beds. Mussels cooperate by attaching themselves to neighbours via byssal threads, thereby providing mutual protection. Using an individual-based model for mussel bed formation, we first demonstrate that the spatial pattern and the corresponding number of neighbours strongly depends on the movement strategies of the mussels. With an evolutionary model, we then show that this has important implications for the evolution of cooperation, since the evolved level of cooperativeness (the number of byssus threads produced) strongly depends on the number of neighbours and on the harshness and variability of the environment. Our results suggest that spatial aggregation, abundantly found in self-organized ecosystems, may promote the evolution of cooperation.     

The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells

We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins alpha 2 beta 1, and alpha 3 beta 1 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-alpha 3 beta 1 mAb, P1F2, localized alpha 3 beta 1 in FAs on purified laminin greater than fibronectin/collagen, indicating that laminin was the best exogeneous ligand for alpha 3 beta 1. However, in long term culture, alpha 3 beta 1 preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-alpha 3 beta 1, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of alpha 3 beta 1 with the secreted laminin-containing ECM was primarily responsible for HFK adhesion in long term culture. (b) In FA assays, alpha 2 beta 1 localized in FAs conincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-alpha 2 beta 1 inhibited initial HFK adhesion to both laminin and collagen. Thus, alpha 2 beta 1 contributes to initial HFK adhesion to laminin but alpha 3 beta 1 is primarily responsible for long-term HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2(+)-induced aggregation of HFKs resulted in relocation of alpha 2 beta 1 and alpha 3 beta 1 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-alpha 3 beta 1 (P1B5) and a new anti-beta 1 mAb (P4C10). Thus, interaction of alpha 3 beta 1 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2(+)-induced HFK aggregation. (d) It is suggested that interaction of alpha 3 beta 1 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of alpha 3 beta 1 in basal cell adhesion to the BMZ in skin. Further, relocation of alpha 2 beta 1 and alpha 3 beta 1 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.     

A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion

The selectin family of adhesion molecules mediates the initial interactions of leukocytes with endothelium. The extracellular region of each selectin contains an amino-terminal C-type lectin domain, followed by an EGF-like domain and multiple short consensus repeat units (SCR). Previous studies have indirectly suggested a role for each of the extracellular domains of the selectins in cell adhesion. In this study, a panel of chimeric selectins created by exchange of domains between L- and P-selectin was used to directly examine the role of the extracellular domains in cell adhesion. Exchange of only the lectin domains between L- and P-selectin conferred the adhesive and ligand recognition functions of the lectin domain of the parent molecule. However, chimeric selectins which contained both the lectin domain of L- selectin and the EGF-like domain of P-selectin exhibited dual ligand- binding specificity. These chimeric proteins supported adhesion both to myeloid cells and to high endothelial venules (HEV) of lymph nodes and mesenteric venules in vivo. Exchange of the SCR domains had no detectable effect on receptor function or specificity. Thus, the EGF- like domain of P-selectin may play a direct role in ligand recognition and leukocyte adhesion mediated by P-selectin, with the lectin plus EGF- like domains collectively forming a functional ligand recognition unit.     

Identification of macrophage external membrane proteins and their possible role in cell adhesion

Starch-activated mouse peritoneal macrophages (STpMAC) plated on plastic demonstrate the adhesive properties typical for activated pMAC: attaching as round cells and, within 15 min, spreading out with marginal membrane ruffles. These attached STpMAC were labeled by lactoperoxidase-catalysed 125I surface iodination, sodium dodecyl- sulfate-lysed, and the lysates electrophoresed on polyacrylamide gels which were examined by autoradiography. The STpMAC morphological phenotype correlates with the labeling of a particular protein (195,000, estimated mol wt). Normal pMAC (NpMAC), from unstimulated mice, do not spread and do not display the 195,000 band. Both pMAC band patterns, including the 195,000 band, are relatively resistant to trypsin digestion, as is pMAC adhesion itself trypsin-resistant. Neither class of pMAC exhibits fibronectin (Cell Adhesion Factor, LETS protein) which is a component in the adhesive matrix of cells forming trypsin-sensitive monolayers. When pMAC are tested against antifibronectin antibody, these cells do not give immunofluorescent staining. In summary, two functions in pMAC adhesion, enzyme resistance and the ability to spread, appear related to molecular properties distinctive for pMAC surface protein.     

A role for fibronectin-leucine-rich transmembrane cell-surface proteins in homotypic cell adhesion

The fibronectin-leucine-rich transmembrane (FLRT) family of leucine-rich repeat (LRR) proteins is implicated in fibroblast growth factor (FGF) signalling, early embryonic development and neurite outgrowth. Here, we have analysed whether FLRTs may also function in cell adhesion. We find that FLRT proteins can physically interact and that FLRT-transfected cultured cells sort out from non-transfected cells, suggesting a change in adhesive properties. A similar sorting effect is also observed in Xenopus embryos and tissue aggregates. FLRT-mediated cell sorting is calcium dependent and substrate independent. Deletion analysis indicates that cell sorting requires the LRR domains, which are dispensable for FLRT-mediated FGF signalling. Conversely, sorting is independent of the cytoplasmic domain, which is essential for FLRT-induced signalling. Therefore, FLRT-mediated FGF signal transduction and homotypic cell sorting can be molecularly uncoupled. The results indicate that FLRT proteins have a dual role, promoting FGF signalling and modulating homotypic cell adhesion.     

A role for the E-cadherin cell-cell adhesion molecule during tumor progression of mouse epidermal carcinogenesis

The expression of the cell-cell adhesion molecules E- and P-cadherin has been analyzed in seven mouse epidermal keratinocyte cell lines representative of different stages of epidermal carcinogenesis. An inverse correlation between the amount of E-cadherin protein and tumorigenicity of the cell lines has been found, together with a complete absence of E-cadherin protein and mRNA expression in three carcinoma cell lines (the epithelioid HaCa4 and the fibroblastoid CarB and CarC cells). A similar result has been detected in tumors induced in nude mice by the cell lines, where induction of E-cadherin expression takes place in moderately differentiated squamous cell carcinomas induced by HaCa4 cells, although at much lower levels than in well-differentiated tumors induced by the epithelial PDV or PDVC57 cell lines. Complete absence of E-cadherin expression has been observed in spindle cell carcinomas induced by CarB or CarC cells. P-cadherin protein was detected in all cell lines that exhibit an epithelial (MCA3D, AT5, PDV, and PDVC57) or epithelioid (HaCa4) morphology, as well as in nude mouse tumors, independent of their tumorigenic capabilities. However, complete absence of P-cadherin was observed in the fibroblast-like cells (CarB and CarC) and in spindle cell carcinomas. The introduction of an exogenous E-cadherin cDNA into HaCa4 cells, or reactivation of the endogenous E-cadherin gene, leads to a partial suppression of the tumorigenicity of this highly malignant cell line. These results suggest a role for E-cadherin in the progression to malignancy of mouse epidermal carcinogenesis. They also suggest that the loss of both E- and P-cadherin could be associated to the final stage of carcinogenesis, the development of spindle cell carcinomas.     

Sticking together and sorting things out: adhesion as a force in development

During development it is not sufficient for cells to differentiate properly--they must also become physically grouped into appropriate structures, to form skin on the outside, and blood and muscle on the inside. How does this three-dimensional patterning occur? One classic explanation for this resolution of cells and tissues into distinct three-dimensional structures has been that as cells differentiate, they develop differential adhesive properties, and that these affinity differences allow cells to sort out from one another. This classic hypothesis is being investigated with increasing intensity, as recent work on the Drosophila wing and the vertebrate brain has shown that signalling between tissues is essential for the establishment of differential affinities.     

Segmental and restricted localization pattern of Fras1 in the developing meningeal basement membrane in mouse

The Fras1/Frem family of extracellular matrix proteins consists of Fras1 and its structurally related proteins, Frem1 (Fras1-related extracellular matrix protein 1), Frem2 and Frem3. These are co-localized in embryonic epithelial basement membranes (BMs), where they contribute to epithelial–mesenchymal adhesion. Although Fras1 localization pattern in epithelial BMs has been well defined, it has not yet been comprehensively studied in the central nervous system. Here, we demonstrate the immunohistochemical profile of Fras1 in the developing mouse brain and reveal an exclusively meningeal BM protein deposition. Interestingly, Fras1 displays a segmental localization pattern, which is restricted to certain regions of the meningeal BM. Frem2 protein displays a similar localization pattern, while Frem3 is rather uniformly distributed throughout the meningeal BM. Fras1 and Frem2 proteins are detected in regions of the BM that underlie organizing centers, such as the roof plate (RP) of diencephalon, midbrain and hindbrain, and the RP-derived structures of telencephalon (choroid plexus and hem). Organizing centers exert their activity via the production of bioactive molecules, which are potential Fras1 ligands. The restricted pattern of Fras1 and Frem2 proteins indicates a molecular compartmentalization of the meningeal BM that could reflect, yet unspecified, functional and structural differences.     

ATH1 and KNAT2 proteins act together in regulation of plant inflorescence architecture

The inflorescence of flowering plants is a highly organized structure, not only contributing to plant reproductive processes, but also constituting an important part of the entire plant morphology. Previous studies have revealed that the class-I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP or KNAT1), KNAT2, and KNAT6 play essential roles in inflorescence architecture. Pedicel morphology is known to contribute greatly to inflorescence architecture, and BP negatively regulates KNAT2 and KNAT6 to ensure that pedicels have a normal upward-pointing orientation. These findings indicate that a genetic network exists in controlling pedicel orientation, but how this network functions in the developmental process remains elusive. Here it is reported that the ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1) gene, which belongs to the BELL1-like homeodomain gene family, is a new member participating in regulating pedicel orientation in the class-I KNOX network. In a genetic screening for suppressors of isoginchaku-2D, a gain-of-function ASYMMETRIC LEAVES2 mutant that displays downward-pointing pedicels, a suppressor mutant was obtained. Characterization of this mutant revealed that the mutation corresponds to ATH1. Genetic analysis indicated that ATH1 acts mainly in the KNAT2 pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that ATH1 physically interacts with KNAT2. The data indicate that the ATH1-KNAT2 complex acts redundantly with KNAT6, both of which are negatively regulated by BP during pedicel development.     

The role of endocytosis in regulating L1-mediated adhesion

L1 is a neural cell adhesion molecule critical for neural development. Full-length L1 (L1(FL)) contains an alternatively spliced cytoplasmic sequence, RSLE, which is absent in L1 expressed in nonneuronal cells. The RSLE sequence follows a tyrosine, creating an endocytic motif that allows rapid internalization via clathrin-mediated endocytosis. We hypothesized that L1(FL) would internalize more rapidly than L1 lacking the RSLE sequence (L1(Delta)(RSLE)) and that internalization might regulate L1-mediated adhesion. L1 internalization was measured by immunofluorescence microscopy and by uptake of (125)I-anti-rat-L1 antibody, demonstrating that L1(FL) is internalized 2-3 times faster than L1(Delta)(RSLE). Inhibition of clathrin-mediated endocytosis slowed internalization of L1(FL) but did not affect initial uptake of L1(Delta)(RSLE). To test whether L1 endocytosis regulates L1 adhesion, cell aggregation rates were tested. L1(Delta)(RSLE) cells aggregated two times faster than L1(FL) cells. Inhibition of clathrin-mediated endocytosis increases the aggregation rate of the L1(FL) cells to that of L1(Delta)(RSLE) cells. Our results demonstrate that rapid internalization of L1 dramatically affects L1 adhesion.     

Ninjurin1: a potential adhesion molecule and its role in inflammation and tissue remodeling

Nerve injury induced protein 1, Ninj1 (Ninjurin1) is a cell surface protein that is induced by nerve injury and promotes axonal growth in the peripheral nervous system. However, the function of Ninj1 in the vascular system and central nervous system (CNS) is incompletely understood. Here we review recent studies that have shed further light on the role and regulation of Ninj1 in vascular remodeling and inflammation. Increasing evidence suggests that Ninj1 mediates cell communication and enhances the entry, migration, and activity of leukocytes such as monocytes and macrophages in developmental processes and inflammatory responses. Moreover, our recent studies show that Ninj1 regulates close interaction between leukocytes and vascular endothelial cells in vascular remodeling and inflamed CNS. Additionally, Ninj1 enhances the apoptosis-inducing activity of leukocytes and is cleaved by MMPs, resulting in loss of adhesion during tissue remodeling. The collective data described here show that Ninj1 is required for the entry, adhesion, activation, and movement of leukocytes during tissue remodeling and might be a potential therapeutic target to regulate the adhesion and trafficking of leukocytes in inflammation and leukocyte-mediated diseases such as multiple sclerosis, diabetic retinopathy, and neuropathy.