Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS-based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy-based height measurements strongly suggest that BMs are more than two-fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi-functionally organized, leading to the proposition that BM-sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue-organizing functions and were essential in the evolution of metazoan species.

Abbreviations

AFM: atomic force microscopy
BM: basement membrane
DN: Descemet's membrane
ECM: extracellular matrix
HSPG: heparan sulfate proteoglycan
ILM: inner limiting membrane
LC: lens capsule
TEM: transmission electron microscopy

The current view of basement membranes

Basement membranes (BMs) are extracellular matrix (ECM) sheets that are located at the basal side of every epithelium; they ensheath smooth, cardiac and skeletal muscle fibers and outline Schwann cells and vascular endothelial cells [1-5]. BMs usually require immunostaining (Fig. 1A) or transmission electron microscopy (TEM) for their detection. According to TEM, the standard BM has a thickness of < 100 nm. Exceptions are the several micrometer-thick lens capsule (LC), the tracheal BM and Descemet's membrane.

Details are in the caption following the image — **Figure 1**
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BMs of the human eye. Section of a fetal human eye (A) stained for collagen IV (green) to highlight the the corneal BMs (Co), the ILM, the lens capsule (LC), the vascular BMs (BV) and the BM of Bruch's membrane (star). L, lens; R, retina; VB, vitreous body. Counter stain: Pax 6 (red). DM, LC, vascular BMs and ILM can be isolated. Scanning electron microscopy showed that the ILM (B) and vascular BMs (C) of an adult human eye are free of cellular debris and non-BM ECM. Scale bar: (A) 200 μm; (B, C) 10 μm.

Key for the identification of BM proteins was the discovery that mouse yolk sac tumors produce a BM-typical ECM in gram quantities [6-8]. Analysis of these tumor matrices showed that laminin [9, 10], nidogen/entactin [11, 12], perlecan [13] and collagen IV [14] are large multidomain glycoproteins with a molecular mass of between 150 and 1000 kDa. Their multiple peptide domains promote polymerization and binding to other BM proteins [15, 16]. The laminins comprise a family of cross-shaped, heterotrimeric glycoproteins with over 10 trimer combinations [17]. Collagen IV is composed of six genetically different α chains that assemble into three linear collagen IV heterotrimers [18]. With molecular masses of ~ 150 kDa, Nidogens I and II are the smallest BM glycoproteins [19-21]. Perlecan was considered the only BM heparan sulfate proteoglycan (HSPG) [13] until two more BM-associated HSPGs, agrin and collagen XVIII, were identified [22-25].

The current model, prominently based on protein binding studies and imaging of isolated BM proteins from the mouse tumors, proposes that BMs are composed of two inter-connected networks: a laminin polymer, which is considered to provide the main cell-binding activity of BMs, and a collagen IV network, which is considered as the main stabilizing structure [26, 27].

Mutations of proteins that are irreplaceable for BM assembly cause embryonic death prior to gastrulation [28]; hence, vertebrates cannot develop and survive without BMs. Nonlethal mutations in mice and humans lead to a collection of BM-typical phenotypes. These include vascular defects that are particularly prominent in brain and eyes [29-33]. Common are retinal or cortical ectopias, in which retinal or cortical cells migrate through breaks in the pial or retinal BMs [29-31, 41-44]. The cortical/retinal BM defects are associated with a major loss of Cajal–Retzius cells in the cortex and at least 50% loss of ganglion cells in the retina [30, 31]. Common phenotypes also include defects in kidney [38, 39] and ear development, whereas skin blisters are restricted to mutations of a subset of the epidermal BM proteins [40]. A common phenotype also includes several forms of congenital muscular dystrophy [36, 41-44]. Many of these pathologies often co-exist as syndromes and are particulary damaging during embryonic development when tissues and organs are expanding and BMs are under stress. In particular, these phenotypes suggest an essential role of mechanical stability for BM functions and that the integrity of BMs is particulalry vulnerable in embryonic and neonatal development. Recent data using mutant mice have emerged demonstrating a key role for integrins and dystroglycan, including the carbohydrate side chain of dystroglycan, in the assembly of many BMs. In particular, mice with defective or no receptors have pathologies that are similar to defects in humans and mice with BM protein mutations [45-50].

Limitations of the current BM model

The commonly accepted thickness of BMs of < 100 nm poses major limitations to model the large BM proteins within the confines of BMs. For collagen IVs with a trimer length of 400 nm, a horizontal positioning is conceivable and, in most diagrams of BMs, the collagen IV network is depicted with a flat, chicken-wire configuration. A high-angle shadowing analysis of the amnion BM has demonstrated a more complex arrangement of collagen IV [51], Nevertheless, in almost all current reviews on BMs, the dominant model of the collagen IV network is still that of a flat, interconnected network [52, 53]. A major issue is that collagens are known to have high tensile strength; thus, a flat positioning would make it difficult for BMs to expand, which might cause functional defects during embryogenesis when tissues and organs are growing. The lacking abbility for BM to expand would also be a major issue for highly flexible adult tissues and organs, such as skin, lens, lung and vasculature. Even for laminins with a length of 80 nm, a vertical positioning appears to be problematic, and diagrams of BM structure regularly depict the laminins in an oblique position [52, 53]. AFM data now show that BMs are thicker than previously considered, providing a greater flexibility in modeling the protein distributions and architecture [54, 55]. Importantly, despite the high amount of proteoglycans present [4, 5], the abundance of water and the hydration status of BMs had not been considered previously.

The complete composition and the stoichiometries of proteins in BMs have not been determined until recently. Nevertheless, it was known that the LC and other human BMs have a dominant presence of collagen IV [56]. MS now allows a detailed and semiquantitative protein analysis of the in vivo derived BMs [57, 58].

Although BMs were considered biomechanically as rigid ECM structures, only few measurements with human LCs have been reported. Hydrostatic test chambers or force transducers were used for these tests [59-61]. Both test methods are not applicable for thinner and smaller sized BMs. A more versatile testing method became available with the advent of atomic force microscopy (AFM) [62], which allows to probe very thin and miniature BM samples [55, 56]. AFM also revealed, for the first time, side-specific differences in the biomechanical properties of BMs [63, 64].

As a result of the focus of BM research on short-lived rodents and invertebrata, age-related changes of BMs had been missed. However, data from human ocular tissues showed that many BMs undergo major compositional, structural and biomechanical changes with advancing age [55].

In TEM micrographs, BMs appear symmetrically structured with a central lamina densa, flanked on either side by morphological similar laminae rarae. Recent experiments, mostly with human ocular BMs, have demonstrated that BMs have a bi-functional organization and that the two surfaces differ in their biomechanical properties, their protein composition and their capacity to promote cell adhesion [64].

The main proposed functions for BM-typical HSPGs are to bind growth factors and to connect the laminin polymer with the collagen IV network. However, recent studies showed that HSPGs also regulate the hydration status of BMs, and thereby determine their biomechanical properties, including thickness and stiffness [55, 57].

New data on BM biology

BM isolation

A prerequisite for studying protein composition and biomechanical properties of BMs is the availability of clean BM samples. BMs are usually isolated from BM-rich tissues by obtaining tissue sheets composed of an epithelial cell layer with its underlying BM. The epithelial cells are then dissolved by detergent leaving the detergent-insoluble BMs behind [65-67]. For many BMs, this isolation method is not applicable. The reason is that many BMs are inseparably connected to much more substantial connective tissue layers that also survive detergent treatments. The detergent-based isolation procedure, however, is applicable for several ocular BMs, such as the inner limiting membrane (ILM) (Figs 1A,B and 2A–C) [63, 64, 68], a BM that separates the vitreous from the retina, the LC and Descemet's membrane (DM) [64], a BM of the cornea. Detergent-based isolation procedures also exist for the vascular BMs of the retina (Fig. 1C) [58, 69], the pial BM of the cerebral cortex [65], the glomerular BMs [70] and BMs from skeletal muscle fibers (A. Labilloy & W. Halfter, unpublished data). A major advantage of ILM, DM and LC is that they can be flat-mounted with known orientation, which is a requirement for side-specific testing.

Protein composition of BMs

The composition of BMs had previously been determined by immunohistochemistry [71, 72]. The studies showed that members of the laminin and collagen IV families, nidogen 1 and 2, and the proteoglycans, perlecan, agrin and collagen XVIII are main BM constituents. Special laminins were detected in epidermal BMs (LNα3β3γ2) [40, 73], muscle BMs (LNα2β1γ1) [74] and vascular BMs (LNα4β1γ1) [75, 76]. ColIV α1α1α2 is the most ubiquitous collagen IV member; however, Col IV α3α4α5 is prominent in glomerular BMs [77] and Col IV α5α5α6 is prominent in Bowman's capsule [77].

An unbiased method to determine the protein constituents of BMs is MS. Isolated BMs are deglycosylated and dissolved in a high molar urea/SDS. After gel electrophoresis, the tryptic peptides of the protein bands are analyzed by LC-MS/MS [57, 58]. The list of proteins is comprehensive and the frequency (or the area under) the peptide peaks in the MS spectra provides a semiquantitative measure of individual proteins. The proteomes of the embryonic chick [57] and the adult human ILM, LC and retinal vascular BMs [58] have been resolved so far. Except for FREM and FRAS in embryonic chick ILM, no unexpected ECM proteins have been detected. FREM and FRAS had been identified by analyzing mice and patients with Faser syndrome. Fraser syndrome leads to skin blistering, and eye defects [78]. Independent studies have shown that these proteins are components of early BMs [79]. Minor proteins in human BMs, as detected by MS, include opticin, emilin, biglycan, serpin B4, papillin, matrilin 2, fibulin 5 and 7, hemicentin 1, tissue inhibitor of metalloproteinase-3, fibroblast growth factor 2, transforming growth factor ß1, vascular endothelial growth factor and heparin-binding semaphorin 3B and 3C [58].

Most importantly, MS allowed for determining the likely peptide chain composition of laminins and collagen IVs and the relative quantities of these major BM proteins: the dominant laminins in embryonic chick ILM, for example, are LNα1β2γ1 and LNα5β2γ1. They occur at a ratio of 1 : 1 and are, together with nidogen 1, the most abundant proteins in the embryonic BM. Collagen IV α5α5α6 is the main collagen IV member in embryonic chick ILM and with 3–5% of the total BM protein only a minor constituent [57]. The dominant laminin family member in the adult human BMs that have been analyzed is LNα5β2γ1. However, other laminin chains (α1; α2; α3; α4 and β1) were also detected. The chain compostion for the dominant collagen IV in adult human BMs varied: ColIV α1α1α2 was the most prominent trimer in LC and vascular BMs, whereas ColIV α3α4α5 was most prominent in the adult human ILM. However, ColIV α3α4α5 is also present in LC and vascular BMs, and little ColIV α1α1α2 was found in ILM. The proteomic data were confirmed by immunohistochemistry. Collagen IVs range between 50% and 70% of the total adult human BM protein [58]. The highest ratio of collagen IV relative to noncollagenous proteins was found in LC and the lowest in ILM. The data showed that embryonic and adult BMs have different protein compositions, and that the relative concentration of collagen IV increases with age. There are multiple laminin and collagen IV family members in BMs but, in most cases, one member is more abundant than the others.

A problem with MS analysis of BMs was that, in some cases, the stoichiometry of the laminin and collagen IV chains did not add up. A likely explanation is that proteins may not all fragment to the same extent into the expected tryptic peptides. For example, for the chick ILM, quantities of laminin β1 and β2 did not match the abundance of laminin γ1 and the combined abundance of laminin α5 and α1 [57]. However, the analysis of human BMs resulted in a better fit for all laminin and collagen IV subunits [58]. Most convincingly, the human data showed uniformity for all dominant proteins between the three biological samples that were analyzed for all three types of BMs [58].

Recent experiments show that collagenase, followed by trypsin digestion, dissolves BMs completely. This allows a more simplified, gel-free proteomic analysis, including proteome comparisons of diabetic versus nondiabetic BMs (S. Moes, K. Halfter, P. Jenoe & W. Halfter, unpublished data).

Biomechanical properties of BMs

With the availability of AFM, thickness and stiffness measurements of very thin and miniature BM samples are now possible [54, 55]. AFM measurements are best performed with flat-mounted samples where both sides can be independently assessed. It is of note that the two sides of all so far tested human BMs have different stiffness values, whereby the epithelial sides are by at least a factor of two stiffer than the stromal sides.

AFM measurments of ILMs, LCs and DMs from neonatal mouse, embryonic chick and adult human tissues revealed that BMs have a stiffness in the order of magnitude of kPa (Fig. 6) [54, 55, 63, 64, 80], higher than hyaline cartilage [81] and one to two orders of magnitude greater than cells [82]. For accurate comparison of the biomechanical data, it is of note that AFM stiffness data calculated using the Sneddon model [83] consistently resulted in higher Pa values than those calculated using the Oliver and Pharr model [84].

Force indentation measurements of ILMs from neonatal mice with defects in the glycosylation of dystroglycans showed a greatly reduced stiffness [80], which a clear sign of mechanically altered BMs. Mechanically weakened BMs explain the frequent breaks of the ILM and pial BMs that are characteristic for these mice (Fig. 2A–D) [80, 85]. BM ruptures in brain and retina are found in mice and fish with mutations of almost all major BM proteins, including perlecan [28], laminins [30, 31, 34, 35, 37, 86] and collagen IV [32, 36, 87]. BM breaks were also observed in mice with central nervous system specific mutation of integrins [47, 49], dystroglycan [50] and enzymes involved in the glycosylation of dystroglycan, including LARGE and POMGnT1 (Fig. 2B,C) [41, 42, 80]. The retinal and cortical breaks occur during embryonic develoment and remain in similar frequency and size in the adulthood [80]. Reduced stiffness of BMs provides a straightforward explanation for the pathology following these mutations and emphasizes that mechanical strength is a major factor for BM functions.

These AFM data show that laminin and other noncollagenous BM proteins contribute significantly to BM stability. Previously, the dominant role in BM stability was assigned to collagen IV [26, 27]. However, studies using stem cells derived from POMGnT1 knockout mice showed that the instability of the BMs in these mice resulted from insufficent amounts of laminin, whereas the relative abundance of collagen IV in these BMs remained unchanged [85].

By contrast to previous observations using TEM, quantitative AFM-based height measurements have demonstrated that BMs are more than two-fold thicker [54, 55]. Confocal microscopy and AFM of human LCs showed a similar thickness difference compared to TEM (~ 29 μm versus 13 μm) [[88], (M. Reyes-Lua, P. Oertle, A. Goz, C. A. Meyer, L. Camenzind, M. Loparic, W. Halfter, P.B. Henrich, submitted)]. Because the increased thickness provides a greater flexilbility for arranging the protein polymers within the confines of BMs, it is no longer required for the collagen IV network to exhibit flat and stretched-out configuration as proposed in almost all BM models. Moreover, it is conceivable that the collagen IV network rather exists in a meshed, yet organized configuration that is currently not understood. A meshed collagen IV network would provide BMs with a greater extensibility that is essential for the development of growing tissues and organs constantly undergoing shape changes. Without lateral elasticity, tissues and organs such as brain, blood vessels, lung, retina and skin would burst or break, and consequently leak.

The explanation for the difference in BM thickness obtained by AFM as compared to TEM is most likely a result of the high water content in BMs. For TEM, tissues samples have to be dehydrated. Hence, TEM produces images of BMs without their normally abundant water. The best candidates for water binding are the HSPGs in BMs because they have long carbohydrate side chains (GAGs) that are highly negatively charged [89]. The GAGs bind counter ions and consequently large quantitites of water. Evidence for the water binding capacity of HSPGs in BMs came from experiments comparing the thickness of BMs with and without GAG side chains: when chick [57] and human ILMs [55] were treated with heparitinase to cleave off the GAG side chains, the thickness of the BMs shrank by over 50% (Fig. 3). Controls showed that the enzymes used in these experiments specifically targeted the GAG side chains and left the HSPG core proteins and the non-HSPG proteins in BMs intact. Moreover, these data have shown that water (with 50% of the total mass) is the most prominent component of BMs and that hydration status, thickness and stiffness of BMs are regulated by the GAG side chains of the HSPGs.

Differences in thickness between TEM and AFM-based measurements were detected for all tested BMs. However, a recent study on kidney glomerular BMs using frozen sections and high resolution fluorescent microscopy [90] showed that thickness data are similar to BM measurements obtained by TEM.

Age-dependent changes of BMs

According to TEM, most BMs appear to have a thickness of < 100 nm. This is true for embryonic and neonatal ILMs from chick (Fig. 4A), adult mouse (Fig. 4B) and ILMs from 2 year-old pigs (Fig. 4C). A similar ILM thickness of < 100 nm was assessed for fetal human eyes (Fig. 4D). TEM micrographs of adult human and nonhuman primate retinas, however, showed that the the thickness of the ILM gradually increases with age and reaches up to 2 μm by the age of 80 years (Fig. 4E–G) [56, 69, 91]. Furthermore, the ILM develops indentations at its retinal surface that become more pronounced with age (Fig. 4E,F). The AFM-based thickness values were by a factor of two greater than the TEM thickness measurements (Fig. 4H) [55, 63, 69]. In addition, the AFM data have confirmed the age-related increase in BM thickness.

An age-dependent increase in BM thickness has been reported for the human epidermal BM [92], human glomerular BMs [93], human DM [94], human LC [61] and human vascular BMs [69, 95].

AFM measurements have revealed an age-related increase in human ILMs stiffness, and western blots and proteome analysis showed a steady, age-related increase in the relative concentration of collagen IV in human BMs [55, 92]. These data combined demonstrate that BMs from humans with advanced age differ from infant BMs by being thicker, stiffer and having different protein compositions. Despite their compositional structural and mechanical changes, adult or even aged human BMs retain their cell adhesive and neurite outgrowth promotive activity [5, 64].

Sidedness of BMs

In TEM micrographs, BMs appear with a symmetrical ultrastructure, showing a lamina densa sandwiched in between two similar appearing lamine rarae (Fig. 4A–D). Yet, a recent study on human epidermal BM [96] showed that the laminin polymer is localized at the epithelial side of the BM, and perlecan was proposed to be the connector to the remaining BM matrix. A one-sided exposure of laminin has also been detected for the chick corneal BM [97].

The testing of a variety of human, mouse and chick BMs suggested that an asymmetrical structure might be universal and inherent to all BMs [64]. A prerequisite for the tests was a reliable, a priori distinction of the epithelial from the stromal sides. Key was the discovery that isolated human BM sheets scroll in a side-specific manner (Fig. 5A). The scrolling depends strictly on the epithelial-stromal polarity of the BMs and not the curvature of the tissue from which the BMs were isolated. Most importantly, the scolling allowed to predicably mount the BMs with either the epithelial or stromal side up. To test both sides next to each other, BMs were flat-mounted in a folded configuration (Fig. 5C,E,F). AFM showed that the epithelial, laminin-rich, sides of all tested BMs are between two and four times stiffer than the stromal, collageneous, sides (Fig. 5B) [63, 64]. It was also found that the two sides have different proteins exposed, which allowed for side-specific staining of BM flat mounts (Fig. 5C) and crossections (Fig. 5D,E) [64]. Data showed that laminin is localized at the epithelial sides of all tested BMs. An antibody to the 7S domain of ColIV α3α4α5 [64, 98] served as a marker for the stromal sides. Using domain specific antibodies to ColIV α3α4α5, it was found that the 7S domain of this collagen IV is localized at the stromal side of BMs, whereas its NC1 domain is localized at the epithelial side, co-localizing with laminin. The side-specific staining was determined for adult human ILM, LC, DM and vascular BMs in peripheral nerves.

Functionally most relevant, cell adhesion experiments revealed side-specific cell adhesion: when epithelial cells were plated onto folded BM flat-mounts, cells adhered much stronger to the epithelial than the stromal sides (Fig. 5E,F): in short-term adhesion assays (15–20 min), all tested epithelial cells adhere exclusively to the epithelial side of BMs. With longer incubation (hours to days), stable cell lines populate both sides but with markedly different densities. Primary cells, however, neither attach to the stromal side, nor migrate from the epithelial into the stromal sides even after long (days) incubation times (W. Halfter, unpublished data). The side-specific properties were detected for all tested human BMs. Although a side-specific protein staining was not detected for mouse and embryonic chick ILMs, possibly as a result of small thickness, side-specific cell adhesion was readily detectable for both mouse and embryonic chick ILMs [64]; thus, cell adhesion assays turned out to be the easiest and most universal way to test for BM sidedness. Furthermore, the sidedness of embryonic and neonatal BMs suggests a built-in BM-asymmetry that is established in its initial assembly.

Recent findings show that the side-specificity does not appear to be limited to the BM surfaces but rather that BMs are composed of two distinct sublayers that can be differentiated based on their stiffness. This was testable by investigating LC samples obtained from cataract surgery. The circular lens capsule segments that are obtained during manual capsulotomy have a wedge-shaped edge, allowing AFM-based stiffness testing throughout the entire thickness of the BM (Fig. 6A,B). When the stiffness across the wedge was measured by AFM, it was found that the epithelial third of the LC has a high stiffness, whereas the vitreal/stromal two-third has a lower stiffness (Fig. 6C) (M. Reyes-Lua, P. Oertle, A. Goz, C. A. Meyer, L. Camenzind, M. Loparic, W. Halfter, P.B. Henrich, submitted).

Side-specific properties of BMs were also detected by transplanting human BMs into chick embryos [99]. The results showed that host-derived ECM proteins, such as collagen II and tenascin-C bound exclusively to the stromal side of the grafted human BMs. Furthermore, when transplanted into the spinal cord, neurons, axons and glia cells arranged themselves in direct contact with and along the epithelial side of the implanted BMs, whereas connective tissue layers, often vascularized, established themselves along the stromal side.

Taken together, the data demonstrate that the epithelial side of BMs is characterized by proteins that promote epithelial cell adhesion, cell migration and axon outgrowth, whereby members of the laminin protein family are main candidates to promote this activity. The stromal side of BMs is endowed with proteins or carbohydrate epitopes that are anti-adhesive but bind stromal-typical ECM proteins, and thereby anchor the BMs firmly with the adjacent connective tissues. The bi-functionality also prevents the formation of directly adjacent epithelial cell layers without an intervening connective tissue. The bi-functional organization of BMs is consistent with countless micrographs of BM zones showing a continuous layer of epithelial cells on the epithelial side of BMs and a dense connective tissue-rich ECM of mostly collagen I, II, fibronectin, fibrillin and tenascin on the stromal side. Further evidence of an active role of BMs in the organization of tissues, mainly based on genetic and in vivo data, has been reviewed recently [100].

Summary and open questions

We postulate that the biochemical and mechanical asymmetry of BMs permit the alternating arrangement of epithelia and connective tissues that is present in all metazoan species. Epithelial cell layers are biomechanically weak [82] and require an up to hundred-fold more resistant substrate for support. The surrounding connective tissues provides the vascularization and innervation that is essential for epithelial cell survival. Most data demonstrating BM sidedness and discussed in the present review were obtained by studying human ocular BMs. A caveat is as to what extent the present findings universally apply to all BMs. Exceptions might be highly specialized BMs such as the kidney glomerular BM where a recent high resolution study did not show a bi-layered substructure [90].

New testing methods uncovered previously little researched properties of in vivo derived BMs. For example, MS confirmed that different BMs have different protein compostions and that the protein composition of BMs changes with age. Most significantly, the relative concentration of collagen IV increases from under 10% during embryonigenesis to over 50% in the adult. Biomechanical testing showed that mutations of BM proteins lead to BM instabilities resulting in vascular ruptures, breaks in the pial BM and disruptions in skeletal muscle BMs. Thus, mechanical weaknesses of BMs explain many of the BM-typical pathologies in mutant mice and humans. BMs appear to be two-fold thicker than previously considered, providing a greater freedom of modelling the very large ECM proteins within. Particularly, the flat and stretched-out configuration of the collagen IV network would no longer be the only possible means of arranging this biopolymer.

An open question remains how the asymmetry of BMs is established. The straightforward way is to assume that epithelial cells first establish laminin-dominated BMs. This initial step is followed by the continuous addition of collagen IV at the stromal side, whereby the collagen IV is synthesized by connective tissue cells. Two notions indicate that this proposition needs further in-depth investigation: staining of human BMs with domain-specfic collagen IV antibodies showed that the structural arrangement of collagen IV in BMs appears to be asymmetric and side-specific: the C-terminal NC1 domain co-localizes with the laminin layer at the epithelial side, whereas the N-terminal 7S domain localizes with the stromal side of BMs [64], suggesting a vertical deposition of collagen IV through the entire BMs. Second, cell adhesion assays with embryonic chick and neonatal mouse BMs showed side-specific cell adhesion, indicating that BMs are assembled from the very beginning with an inherent asymmetry [64].

BMs represent barriers and filters through which material diffuses. It is unknown what size and at what rate proteins diffuse through BMs. This question is particularly important for BMs of the vasculature and secretory organs, where filtration rate and size exclusion are key functional elements. Similarly, the mode of the damage-free passage of neutrophils through BMs needs further investigation [101-103]. Studies in Caenorhabditis elegans have recently shown that anchor cells also pass a BM without leaving a detectable breach [104, 105]. The passage of the anchor cell was recorded by time lapse, and the recordings indicated that the intervening BM network is capable of temporarily expanding, allowing the cell to pass, after which the elastic fibrils of the BM contract and close the temporary opening [104, 105]. The fact that cells can pass BMs without structural damage infers a fibrillar struture of BMs that is highly flexible and expandable, which is in agreement with the data discussed in this review.

Recent data show morphological, compositional and functional sidedness of BMs and demonstrate that BMs are structurally more sophisticated than previously considered. The side-specific adhesives functionally connects otherwise incompatible epithelia and connective tissues. Taken together, these new findings call for a renewed effort aiming to investigate, revise and eventually understand the structual compositions of in vivo derived BMs and their influence on the development and maintenance of epithelia.

Acknowledgements

We would like to thank Daphne Asgeirsson for the critical reading of the manuscript.

Author contributions

The experimental work was done by Willi Halfter, Christophe Monnier, Philipp Oertle, Magaly Reyes-Lua, Leon Camenzind, Anatalya Labilloy, Manimalha Balasubramani, Haiyu Hu, Joseph Candiello. Willi Halfter, Berhard Henrich and Marija Plodinec were laboratory heads and contributed in writing the manuscript and designing the experiments.

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Volume282, Issue23

December 2015

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