REVIEW ARTICLE New concepts in biology Willi Halfter1, Philipp Oertle2, Christophe A. Monnier2,*, Leon Camenzind2, Magaly Reyes-Lua1, Huaiyu Hu3, Joseph Candiello4, Anatalia Labilloy5,†, Manimalha Balasubramani6, Paul Bernhard Henrich1 and Marija Plodinec2,7

1 Department of Ophthalmology, University Hospital Basel, Switzerland 2 Biozentrum and the Swiss Nanoscience Institute, University of Basel, Switzerland 3 Department of Neurobiology and Physiology, Upstate University Hospital, SUNY University, Syracuse, NY, USA 4 Department of Bioengeneering, University of Pittsburgh, PA, USA 5 Department of Renal Physiology, University of Pittsburgh, PA, USA 6 Proteomics Core Facility of the University of Pittsburgh, PA, USA 7 Department of Pathology, University Hospital Basel, Switzerland

Keywords Basement membranes (BMs) are thin sheets of that basal lamina; basement membrane; outline epithelia, muscle fibers, blood vessels and peripheral nerves. The biomechanical properties; IV; current view of BM structure and functions is based mainly on transmis- ; membrane asymmetry; nidogen; sion electron microscopy imaging, in vitro binding assays, and phe- perlecan notype analysis of human patients, mutant mice and invertebrata. Correspondence Recently, MS-based protein analysis, biomechanical testing and cell adhe- W. Halfter, Department of Ophthalmology, sion assays with in vivo derived BMs have led to new and unexpected University Hospital Basel, Mittlere insights. Proteomic analysis combined with ultrastructural studies showed Strasse 91, 4031 Basel, Switzerland that many BMs undergo compositional and structural changes with Fax: +41 61 267 21 09 advancing age. Atomic force microscopy measurements in combination Tel: +49 7624 982528 with phenotype analysis have revealed an altered mechanical stiffness that E-mail: [email protected] M. Plodinec, Department of Pathology, correlates with specific BM pathologies in mutant mice and human University Hospital Basel, patients. Atomic force microscopy-based height measurements strongly Schonbeinstrasse€ 40, CH-4031 Basel, suggest that BMs are more than two-fold thicker than previously esti- Switzerland mated, providing greater freedom for modelling the large protein polymers Fax: +41 61 267 21 09 within BMs. In addition, data gathered using BMs extracted from mutant Tel: +41 267 22 60 mice showed that laminin has a crucial role in BM stability. Finally, recent E-mail: [email protected] evidence demonstrate that BMs are bi-functionally organized, leading to Present addresses: the proposition that BM-sidedness contributes to the alternating epithelial *Adolphe Merkle Institute, University of and stromal tissue arrangements that are found in all metazoan species. Fribourg, Fribourg, Switzerland We propose that BMs are ancient structures with tissue-organizing func- †Cincinnati Children’s Hospital Medical tions and were essential in the evolution of metazoan species. Center, Cincinnati, OH, USA

(Received 22 April 2015, revised 13 July 2015, accepted 18 August 2015) doi:10.1111/febs.13495

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.

4466 FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. W. Halfter et al. New concepts in basement membrane biology

The current view of basement Mutations of that are irreplaceable for BM membranes assembly cause embryonic death prior to gastrulation [28]; hence, vertebrates cannot develop and survive Basement membranes (BMs) are extracellular matrix without BMs. Nonlethal mutations in mice and (ECM) sheets that are located at the basal side of humans lead to a collection of BM-typical phenotypes. every epithelium; they ensheath smooth, cardiac and These include vascular defects that are particularly skeletal muscle fibers and outline Schwann cells and prominent in brain and eyes [29–33]. Common are reti- – vascular endothelial cells [1 5]. BMs usually require nal or cortical ectopias, in which retinal or cortical immunostaining (Fig. 1A) or transmission electron cells migrate through breaks in the pial or retinal BMs microscopy (TEM) for their detection. According to [29–31,34–37]. The cortical/retinal BM defects are < TEM, the standard BM has a thickness of 100 nm. associated with a major loss of Cajal–Retzius cells in Exceptions are the several micrometer-thick lens the cortex and at least 50% loss of ganglion cells in capsule (LC), the tracheal BM and Descemet’s mem- the retina [30,31]. Common phenotypes also include brane. defects in kidney [38,39] and ear development, whereas Key for the identification of BM proteins was the skin blisters are restricted to mutations of a subset of discovery that mouse yolk sac tumors produce a BM- the epidermal BM proteins [40]. A common phenotype – typical ECM in gram quantities [6 8]. Analysis of also includes several forms of congenital muscular these tumor matrices showed that laminin [9,10], nido- dystrophy [36,41–44]. Many of these pathologies often gen/entactin [11,12], perlecan [13] and collagen IV [14] co-exist as syndromes and are particulary damaging are large multidomain glycoproteins with a molecular during embryonic development when tissues and organs mass of between 150 and 1000 kDa. Their multiple are expanding and BMs are under stress. In particular, peptide domains promote polymerization and binding these phenotypes suggest an essential role of mechani- to other BM proteins [15,16]. The comprise a cal stability for BM functions and that the integrity of family of cross-shaped, heterotrimeric glycoproteins BMs is particulalry vulnerable in embryonic and neona- with over 10 trimer combinations [17]. Collagen IV is tal development. Recent data using mutant mice have a composed of six genetically different chains that emerged demonstrating a key role for integrins and assemble into three linear collagen IV heterotrimers dystroglycan, including the carbohydrate side chain of ~ [18]. With molecular masses of 150 kDa, Nidogens I dystroglycan, in the assembly of many BMs. In particu- – and II are the smallest BM glycoproteins [19 21]. Per- lar, mice with defective or no receptors have patholo- lecan was considered the only BM heparan sulfate pro- gies that are similar to defects in humans and mice with teoglycan (HSPG) [13] until two more BM-associated BM protein mutations [45–50]. HSPGs, agrin and collagen XVIII, were identified [22–25]. The current model, prominently based on protein Limitations of the current BM model binding studies and imaging of isolated BM proteins The commonly accepted thickness of BMs of from the mouse tumors, proposes that BMs are com- < 100 nm poses major limitations to model the large posed of two inter-connected networks: a laminin BM proteins within the confines of BMs. For collagen polymer, which is considered to provide the main cell- IVs with a trimer length of 400 nm, a horizontal posi- binding activity of BMs, and a collagen IV network, tioning is conceivable and, in most diagrams of BMs, which is considered as the main stabilizing structure the collagen IV network is depicted with a flat, [26,27].

ABC

Fig. 1. 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 lm; (B, C) 10 lm.

FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 4467 Federation of European Biochemical Societies. New concepts in basement membrane biology W. Halfter et al. chicken-wire configuration. A high-angle shadowing biomechanical properties, their protein composition analysis of the amnion BM has demonstrated a more and their capacity to promote cell adhesion [64]. complex arrangement of collagen IV [51], Nevertheless, The main proposed functions for BM-typical in almost all current reviews on BMs, the dominant HSPGs are to bind growth factors and to connect the model of the collagen IV network is still that of a flat, laminin polymer with the collagen IV network. How- interconnected network [52,53]. A major issue is that ever, recent studies showed that HSPGs also regulate are known to have high tensile strength; the hydration status of BMs, and thereby determine thus, a flat positioning would make it difficult for their biomechanical properties, including thickness and BMs to expand, which might cause functional defects stiffness [55,57]. during embryogenesis when tissues and organs are growing. The lacking abbility for BM to expand would New data on BM biology also be a major issue for highly flexible adult tissues and organs, such as skin, lens, lung and vasculature. BM isolation Even for laminins with a length of 80 nm, a vertical positioning appears to be problematic, and diagrams A prerequisite for studying protein composition and of BM structure regularly depict the laminins in an biomechanical properties of BMs is the availability of oblique position [52,53]. AFM data now show that clean BM samples. BMs are usually isolated from BM- BMs are thicker than previously considered, providing rich tissues by obtaining tissue sheets composed of an a greater flexibility in modeling the protein distribu- epithelial cell layer with its underlying BM. The epithe- tions and architecture [54,55]. Importantly, despite the lial cells are then dissolved by detergent leaving the high amount of proteoglycans present [4,5], the abun- detergent-insoluble BMs behind [65–67]. For many dance of water and the hydration status of BMs had BMs, this isolation method is not applicable. The rea- not been considered previously. son is that many BMs are inseparably connected to The complete composition and the stoichiometries much more substantial connective tissue layers that of proteins in BMs have not been determined until also survive detergent treatments. The detergent-based recently. Nevertheless, it was known that the LC and isolation procedure, however, is applicable for several other human BMs have a dominant presence of colla- ocular BMs, such as the inner limiting membrane gen IV [56]. MS now allows a detailed and semiquanti- (ILM) (Figs 1A,B and 2A–C) [63,64,68], a BM that tative protein analysis of the in vivo derived BMs separates the vitreous from the retina, the LC and [57,58]. Descemet’s membrane (DM) [64], a BM of the cornea. Although BMs were considered biomechanically as Detergent-based isolation procedures also exist for the rigid ECM structures, only few measurements with vascular BMs of the retina (Fig. 1C) [58,69], the pial human LCs have been reported. Hydrostatic test BM of the cerebral cortex [65], the glomerular BMs chambers or force transducers were used for these tests [70] and BMs from skeletal muscle fibers (A. Labilloy [59–61]. Both test methods are not applicable for thin- & W. Halfter, unpublished data). A major advantage ner and smaller sized BMs. A more versatile testing of ILM, DM and LC is that they can be flat-mounted method became available with the advent of atomic with known orientation, which is a requirement for force microscopy (AFM) [62], which allows to probe side-specific testing. very thin and miniature BM samples [55,56]. AFM also revealed, for the first time, side-specific differences Protein composition of BMs in the biomechanical properties of BMs [63,64]. As a result of the focus of BM research on short- The composition of BMs had previously been deter- lived rodents and invertebrata, age-related changes of mined by immunohistochemistry [71,72]. The studies BMs had been missed. However, data from human showed that members of the laminin and collagen IV ocular tissues showed that many BMs undergo major families, nidogen 1 and 2, and the proteoglycans, per- compositional, structural and biomechanical changes lecan, agrin and collagen XVIII are main BM con- with advancing age [55]. stituents. Special laminins were detected in epidermal In TEM micrographs, BMs appear symmetrically BMs (LNa3b3c2) [40,73], muscle BMs (LNa2b1c1) structured with a central lamina densa, flanked on [74] and vascular BMs (LNa4b1c1) [75,76]. ColIV either side by morphological similar laminae rarae. a1a1a2 is the most ubiquitous collagen IV member; Recent experiments, mostly with human ocular BMs, however, Col IV a3a4a5 is prominent in glomerular have demonstrated that BMs have a bi-functional BMs [77] and Col IV a5a5a6 is prominent in Bow- organization and that the two surfaces differ in their man’s capsule [77].

4468 FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. W. Halfter et al. New concepts in basement membrane biology

A BC

DE

Fig. 2. Immunofluorescent images of flat-mounted ILMs from a neonatal wild-type control mouse (A) and from mutant mice with deletions of LARGE (B) and POMGnT1 (C). LARGE and POMGnT1 are enzymes that participate in the glycosylation of dystroglycan. The collagen IV staining of the wild-type control ILM shows a continous BM (A). The diagonal scratch was made to measure the height of the ILM from a defined edge. ILMs from the mutant mice (B, C) are perforated at many sites as a result of the fragility of the BM. Representative force curves of the ILMs (D) show that the wild-type ILM (WT) has a greater stiffness than the ILMs from the POMGnT1 and the LARGE mice. The stiffness was calculated from the intial slope of the force curves with an indentation into the BMs for the first 25 nm. The greater the slope, the stiffer the material. AFM height profiles show that the LARGE ILM is only slightly thinner than the ILM from the wild type ILM (E). Scale bar: (A–C) 25 lm.

An unbiased method to determine the protein con- Most importantly, MS allowed for determining the stituents of BMs is MS. Isolated BMs are deglycosy- likely peptide chain composition of laminins and colla- lated and dissolved in a high molar urea/SDS. After gen IVs and the relative quantities of these major BM gel electrophoresis, the tryptic peptides of the protein proteins: the dominant laminins in embryonic chick bands are analyzed by LC-MS/MS [57,58]. The list of ILM, for example, are LNa1b2c1 and LNa5b2c1. proteins is comprehensive and the frequency (or the They occur at a ratio of 1 : 1 and are, together with area under) the peptide peaks in the MS spectra pro- nidogen 1, the most abundant proteins in the embry- vides a semiquantitative measure of individual pro- onic BM. Collagen IV a5a5a6 is the main collagen IV teins. The proteomes of the embryonic chick [57] and member in embryonic chick ILM and with 3–5% of the adult human ILM, LC and retinal vascular BMs the total BM protein only a minor constituent [57]. [58] have been resolved so far. Except for FREM and The dominant laminin family member in the adult FRAS in embryonic chick ILM, no unexpected ECM human BMs that have been analyzed is LNa5b2c1. proteins have been detected. FREM and FRAS had However, other laminin chains (a1; a2; a3; a4 and b1) been identified by analyzing mice and patients with were also detected. The chain compostion for the dom- Faser syndrome. Fraser syndrome leads to skin blister- inant collagen IV in adult human BMs varied: ColIV ing, and eye defects [78]. Independent studies have a1a1a2 was the most prominent trimer in LC and vas- shown that these proteins are components of early cular BMs, whereas ColIV a3a4a5 was most promi- BMs [79]. Minor proteins in human BMs, as detected nent in the adult human ILM. However, ColIV by MS, include opticin, emilin, biglycan, serpin B4, a3a4a5 is also present in LC and vascular BMs, and papillin, matrilin 2, fibulin 5 and 7, hemicentin 1, tis- little ColIV a1a1a2 was found in ILM. The proteomic sue inhibitor of metalloproteinase-3, fibroblast growth data were confirmed by immunohistochemistry. Colla- factor 2, transforming growth factor ß1, vascular gen IVs range between 50% and 70% of the total endothelial growth factor and heparin-binding sema- adult human BM protein [58]. The highest ratio of col- phorin 3B and 3C [58]. lagen IV relative to noncollagenous proteins was found

FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 4469 Federation of European Biochemical Societies. New concepts in basement membrane biology W. Halfter et al. in LC and the lowest in ILM. The data showed that brain and retina are found in mice and fish with muta- embryonic and adult BMs have different protein com- tions of almost all major BM proteins, including per- positions, and that the relative concentration of colla- lecan [28], laminins [30,31,34,35,37,86] and collagen IV gen IV increases with age. There are multiple laminin [32,36,87]. BM breaks were also observed in mice with and collagen IV family members in BMs but, in most central nervous system specific mutation of integrins cases, one member is more abundant than the others. [47,49], dystroglycan [50] and enzymes involved in the A problem with MS analysis of BMs was that, in glycosylation of dystroglycan, including LARGE and some cases, the stoichiometry of the laminin and colla- POMGnT1 (Fig. 2B,C) [41,42,80]. The retinal and cor- gen IV chains did not add up. A likely explanation is tical breaks occur during embryonic develoment and that proteins may not all fragment to the same extent remain in similar frequency and size in the adulthood into the expected tryptic peptides. For example, for [80]. Reduced stiffness of BMs provides a straightfor- the chick ILM, quantities of laminin b1 and b2 did ward explanation for the pathology following these not match the abundance of laminin c1 and the com- mutations and emphasizes that mechanical strength is bined abundance of laminin a5 and a1 [57]. However, a major factor for BM functions. the analysis of human BMs resulted in a better fit for These AFM data show that laminin and other non- all laminin and collagen IV subunits [58]. Most con- collagenous BM proteins contribute significantly to vincingly, the human data showed uniformity for all BM stability. Previously, the dominant role in BM sta- dominant proteins between the three biological sam- bility was assigned to collagen IV [26,27]. However, ples that were analyzed for all three types of BMs [58]. studies using stem cells derived from POMGnT1 Recent experiments show that collagenase, followed knockout mice showed that the instability of the BMs by trypsin digestion, dissolves BMs completely. This in these mice resulted from insufficent amounts of allows a more simplified, gel-free proteomic analysis, laminin, whereas the relative abundance of collagen IV including proteome comparisons of diabetic versus in these BMs remained unchanged [85]. nondiabetic BMs (S. Moes, K. Halfter, P. Jenoe & W. By contrast to previous observations using TEM, Halfter, unpublished data). quantitative AFM-based height measurements have demonstrated that BMs are more than two-fold thicker [54,55]. Confocal microscopy and AFM of human LCs Biomechanical properties of BMs showed a similar thickness difference compared to With the availability of AFM, thickness and stiffness TEM (~ 29 lm versus 13 lm) [88, (M. Reyes-Lua, P. measurements of very thin and miniature BM samples Oertle, A. Goz, C. A. Meyer, L. Camenzind, M. Lopa- are now possible [54,55]. AFM measurements are best ric, W. Halfter, P.B. Henrich, submitted)]. Because the performed with flat-mounted samples where both sides increased thickness provides a greater flexilbility for can be independently assessed. It is of note that the arranging the protein polymers within the confines of two sides of all so far tested human BMs have differ- BMs, it is no longer required for the collagen IV net- ent stiffness values, whereby the epithelial sides are by work to exhibit flat and stretched-out configuration as at least a factor of two stiffer than the stromal sides. proposed in almost all BM models. Moreover, it is AFM measurments of ILMs, LCs and DMs from conceivable that the collagen IV network rather exists neonatal mouse, embryonic chick and adult human tis- in a meshed, yet organized configuration that is cur- sues revealed that BMs have a stiffness in the order of rently not understood. A meshed collagen IV network magnitude of kPa (Fig. 6) [54,55,63,64,80], higher than would provide BMs with a greater extensibility that is hyaline cartilage [81] and one to two orders of magni- essential for the development of growing tissues and tude greater than cells [82]. For accurate comparison organs constantly undergoing shape changes. Without of the biomechanical data, it is of note that AFM stiff- lateral elasticity, tissues and organs such as brain, ness data calculated using the Sneddon model [83] con- blood vessels, lung, retina and skin would burst or sistently resulted in higher Pa values than those break, and consequently leak. calculated using the Oliver and Pharr model [84]. The explanation for the difference in BM thickness Force indentation measurements of ILMs from obtained by AFM as compared to TEM is most likely neonatal mice with defects in the glycosylation of dys- a result of the high water content in BMs. For TEM, troglycans showed a greatly reduced stiffness [80], tissues samples have to be dehydrated. Hence, TEM which a clear sign of mechanically altered BMs. produces images of BMs without their normally abun- Mechanically weakened BMs explain the frequent dant water. The best candidates for water binding are breaks of the ILM and pial BMs that are characteristic the HSPGs in BMs because they have long carbohy- for these mice (Fig. 2A–D) [80,85]. BM ruptures in drate side chains (GAGs) that are highly negatively

4470 FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. W. Halfter et al. New concepts in basement membrane biology charged [89]. The GAGs bind counter ions and conse- AFM-based thickness values were by a factor of two quently large quantitites of water. Evidence for the greater than the TEM thickness measurements water binding capacity of HSPGs in BMs came from (Fig. 4H) [55,63,69]. In addition, the AFM data have experiments comparing the thickness of BMs with and confirmed the age-related increase in BM thickness. without GAG side chains: when chick [57] and human An age-dependent increase in BM thickness has ILMs [55] were treated with heparitinase to cleave off been reported for the human epidermal BM [92], the GAG side chains, the thickness of the BMs shrank human glomerular BMs [93], human DM [94], human by over 50% (Fig. 3). Controls showed that the LC [61] and human vascular BMs [69,95]. enzymes used in these experiments specifically targeted AFM measurements have revealed an age- the GAG side chains and left the HSPG core proteins related increase in human ILMs stiffness, and west- and the non-HSPG proteins in BMs intact. Moreover, ern blots and proteome analysis showed a steady, these data have shown that water (with 50% of the age-related increase in the relative concentration of total mass) is the most prominent component of BMs collagen IV in human BMs [55,92]. These data com- and that hydration status, thickness and stiffness of bined demonstrate that BMs from humans with BMs are regulated by the GAG side chains of the advanced age differ from infant BMs by being thicker, HSPGs. stiffer and having different protein compositions. Differences in thickness between TEM and AFM- Despite their compositional structural and mechanical based measurements were detected for all tested BMs. changes, adult or even aged human BMs retain their However, a recent study on kidney glomerular BMs cell adhesive and neurite outgrowth promotive activity using frozen sections and high resolution fluorescent [5,64]. microscopy [90] showed that thickness data are similar to BM measurements obtained by TEM. Sidedness of BMs In TEM micrographs, BMs appear with a symmetrical Age-dependent changes of BMs ultrastructure, showing a lamina densa sandwiched in According to TEM, most BMs appear to have a thick- between two similar appearing lamine rarae (Fig. 4A– ness of < 100 nm. This is true for embryonic and D). Yet, a recent study on human epidermal BM [96] neonatal ILMs from chick (Fig. 4A), adult mouse showed that the laminin polymer is localized at the (Fig. 4B) and ILMs from 2 year-old pigs (Fig. 4C). A epithelial side of the BM, and perlecan was proposed similar ILM thickness of < 100 nm was assessed for to be the connector to the remaining BM matrix. A fetal human eyes (Fig. 4D). TEM micrographs of one-sided exposure of laminin has also been detected adult human and nonhuman primate retinas, however, for the chick corneal BM [97]. showed that the the thickness of the ILM gradually The testing of a variety of human, mouse and chick increases with age and reaches up to 2 lm by the age BMs suggested that an asymmetrical structure might of 80 years (Fig. 4E–G) [56,69,91]. Furthermore, the be universal and inherent to all BMs [64]. A prerequi- ILM develops indentations at its retinal surface that site for the tests was a reliable, a priori distinction of become more pronounced with age (Fig. 4E,F). The the epithelial from the stromal sides. Key was the dis-

AB

Fig. 3. AFM-based thickness and stiffness measurements of embryonic chick and adult human ILMs with and without HS-GAG chains. Representative thickness traces of a control chick ILM (Cont) and a chick ILM after heparitinase treatment (Hep) are shown in (A). After deglycosylation, the ILM shrank by 50%. Thickness data from human ILMs of different ages are shown in (B): in all cases (C), heparitinase (H) led to a decrease in BM thickness relative to controls (C).

FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 4471 Federation of European Biochemical Societies. New concepts in basement membrane biology W. Halfter et al.

AB

CD

EF Fig. 4. Age-related increase in human BM thickness. TEM micrographs show the ILM of a chick embryos (A), a neontal mouse (B) a 2-year old pig (C), a fetal human eye (D), a 17-year-old (E) and a 62- year-old human donor (F). The fetal human GH ILM has the same ultrastructure as the chick, mouse and pig ILM with a thickness of < 100 nm. With advancing age, the human ILM increased in thickness and developed irregular extensions at its retinal surface (E, F). The age-dependent increase in ILM thickness, based on TEM only, is shown in (G). The plot in (H) compared the age-dependent increase in ILM thickness based on AFM and TEM measurements. Scale bar: (A–F) 0.5 lm. covery that isolated human BM sheets scroll in a side- epithelial cells were plated onto folded BM flat- specific manner (Fig. 5A). The scrolling depends mounts, cells adhered much stronger to the epithelial strictly on the epithelial-stromal polarity of the BMs than the stromal sides (Fig. 5E,F): in short-term and not the curvature of the tissue from which the adhesion assays (15–20 min), all tested epithelial cells BMs were isolated. Most importantly, the scolling adhere exclusively to the epithelial side of BMs. With allowed to predicably mount the BMs with either the longer incubation (hours to days), stable cell lines epithelial or stromal side up. To test both sides next to populate both sides but with markedly different densi- each other, BMs were flat-mounted in a folded config- ties. Primary cells, however, neither attach to the stro- uration (Fig. 5C,E,F). AFM showed that the epithe- mal side, nor migrate from the epithelial into the lial, laminin-rich, sides of all tested BMs are between stromal sides even after long (days) incubation times two and four times stiffer than the stromal, collage- (W. Halfter, unpublished data). The side-specific neous, sides (Fig. 5B) [63,64]. It was also found that properties were detected for all tested human BMs. the two sides have different proteins exposed, which Although a side-specific protein staining was not allowed for side-specific staining of BM flat mounts detected for mouse and embryonic chick ILMs, possi- (Fig. 5C) and crossections (Fig. 5D,E) [64]. Data bly as a result of small thickness, side-specific cell showed that laminin is localized at the epithelial sides adhesion was readily detectable for both mouse and of all tested BMs. An antibody to the 7S domain of embryonic chick ILMs [64]; thus, cell adhesion assays ColIV a3a4a5 [64,98] served as a marker for the stro- turned out to be the easiest and most universal way mal sides. Using domain specific antibodies to ColIV to test for BM sidedness. Furthermore, the sidedness a3a4a5, it was found that the 7S domain of this colla- of embryonic and neonatal BMs suggests a built-in gen IV is localized at the stromal side of BMs, whereas BM-asymmetry that is established in its initial its NC1 domain is localized at the epithelial side, co- assembly. localizing with laminin. The side-specific staining was Recent findings show that the side-specificity does determined for adult human ILM, LC, DM and vascu- not appear to be limited to the BM surfaces but rather lar BMs in peripheral nerves. that BMs are composed of two distinct sublayers that Functionally most relevant, cell adhesion experi- can be differentiated based on their stiffness. This was ments revealed side-specific cell adhesion: when testable by investigating LC samples obtained from

4472 FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. W. Halfter et al. New concepts in basement membrane biology

A B C Fig. 5. Sidedness of human BMs. A free- floating segment of adult human ILM, stained for pan-collagen IV, is shown in (A). The retinal/epithelial (R) side of the scrolled ILM is facing outward and the vitreal/stromal side (V) facing inward (A). AFM measurments (B) showed that the retinal side of the ILM is approximately twice stiffer than the vitreal side. A flat- mounted and folded human ILM was stained for laminin (C; red) to label the DE retinal (epithelial) side. Staining for the 7S domain of ColIV a3a4a5 (B; green) was used to label the vitreal (stromal) side. The same side-specific localization of protein/ peptide domains was detected in crossections of human retina (D) and capillaries of peripheral nerve (E) (En, F G endothelial side; St, stromal side). When rabbit corneal epithelial cells were plated onto a folded human LC, the cells adhered to the epithelial side of the BM (E) and not to the vitreal/stromal side (V) as shown by dark field (F). The side-specific cell adhesion was confirmed (G) by staining of the vitreal side (V) of the same LC sample (boxed) using an antibody to the 7S domain of ColIV a3a4a5 (red). The adherent cells were labeled with SytoxGreen (G). Scale bars: (B) 100 lm; (C) 0.5 lm; (D) 200 lm; (E) 100 lm. cataract surgery. The circular lens capsule segments promote epithelial cell adhesion, cell migration and that are obtained during manual capsulotomy have a axon outgrowth, whereby members of the laminin pro- wedge-shaped edge, allowing AFM-based stiffness test- tein family are main candidates to promote this activ- ing throughout the entire thickness of the BM ity. The stromal side of BMs is endowed with proteins (Fig. 6A,B). When the stiffness across the wedge was or carbohydrate epitopes that are anti-adhesive but measured by AFM, it was found that the epithelial bind stromal-typical ECM proteins, and thereby third of the LC has a high stiffness, whereas the vit- anchor the BMs firmly with the adjacent connective real/stromal two-third has a lower stiffness (Fig. 6C) tissues. The bi-functionality also prevents the forma- (M. Reyes-Lua, P. Oertle, A. Goz, C. A. Meyer, L. tion of directly adjacent epithelial cell layers without Camenzind, M. Loparic, W. Halfter, P.B. Henrich, an intervening connective tissue. The bi-functional submitted). organization of BMs is consistent with countless Side-specific properties of BMs were also detected by micrographs of BM zones showing a continuous layer transplanting human BMs into chick embryos [99]. The of epithelial cells on the epithelial side of BMs and a results showed that host-derived ECM proteins, such as dense connective tissue-rich ECM of mostly collagen I, collagen II and -C bound exclusively to the II, fibronectin, fibrillin and tenascin on the stromal stromal side of the grafted human BMs. Furthermore, side. Further evidence of an active role of BMs in the when transplanted into the spinal cord, neurons, axons organization of tissues, mainly based on genetic and and glia cells arranged themselves in direct contact with in vivo data, has been reviewed recently [100]. and along the epithelial side of the implanted BMs, whereas connective tissue layers, often vascularized, Summary and open questions established themselves along the stromal side. Taken together, the data demonstrate that the We postulate that the biochemical and mechanical epithelial side of BMs is characterized by proteins that asymmetry of BMs permit the alternating arrangement

FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 4473 Federation of European Biochemical Societies. New concepts in basement membrane biology W. Halfter et al.

A B

Fig. 6. Side-specific AFM-testing of the lens capsule. During cataract surgery, the C anterior lens capsule is opened, and a circular segment of th elens capsule is removed to access the interior of the lens. The rim of the removed lens capsule segment is wedge-shaped as shown by light microscopy (A). The width of the wedge is ~ 70 lm and has an angle of 30o. The wedge-shaped rim was confirmed by scanning electron microscopy (B). The AFM stiffness map accross the rims showed a tough epithelial third (Ep) (red in C) and a much softer vitreal/stromal tow-third (St) (blue in C). The border between high stiffness (red) and low stiffness (blue) is close to the middle of the lens capsule, indicating that the BM is composed of two biomechanically distinctive sublayers. of epithelia and connective tissues that is present in weaknesses of BMs explain many of the BM-typical all metazoan species. Epithelial cell layers are biome- pathologies in mutant mice and humans. BMs appear chanically weak [82] and require an up to hundred- to be two-fold thicker than previously considered, pro- fold more resistant substrate for support. The sur- viding a greater freedom of modelling the very large rounding connective tissues provides the vasculariza- ECM proteins within. Particularly, the flat and tion and innervation that is essential for epithelial cell stretched-out configuration of the collagen IV network survival. Most data demonstrating BM sidedness and would no longer be the only possible means of arrang- discussed in the present review were obtained by ing this biopolymer. studying human ocular BMs. A caveat is as to what An open question remains how the asymmetry of extent the present findings universally apply to all BMs is established. The straightforward way is to BMs. Exceptions might be highly specialized BMs assume that epithelial cells first establish laminin-domi- such as the kidney glomerular BM where a recent nated BMs. This initial step is followed by the contin- high resolution study did not show a bi-layered sub- uous addition of collagen IV at the stromal side, structure [90]. whereby the collagen IV is synthesized by connective New testing methods uncovered previously little tissue cells. Two notions indicate that this proposition researched properties of in vivo derived BMs. For needs further in-depth investigation: staining of human example, MS confirmed that different BMs have dif- BMs with domain-specfic collagen IV antibodies ferent protein compostions and that the protein com- showed that the structural arrangement of collagen IV position of BMs changes with age. Most significantly, in BMs appears to be asymmetric and side-specific: the the relative concentration of collagen IV increases C-terminal NC1 domain co-localizes with the laminin from under 10% during embryonigenesis to over 50% layer at the epithelial side, whereas the N-terminal 7S in the adult. Biomechanical testing showed that muta- domain localizes with the stromal side of BMs [64], tions of BM proteins lead to BM instabilities resulting suggesting a vertical deposition of collagen IV through in vascular ruptures, breaks in the pial BM and dis- the entire BMs. Second, cell adhesion assays with ruptions in skeletal muscle BMs. Thus, mechanical embryonic chick and neonatal mouse BMs showed

4474 FEBS Journal 282 (2015) 4466–4479 ª 2015 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. W. Halfter et al. New concepts in basement membrane biology side-specific cell adhesion, indicating that BMs are 2 Vracko R (1974) Basal lamina scaffold-anatomy and assembled from the very beginning with an inherent significance for maintenance of orderly tissue structure. asymmetry [64]. Am J Pathol 77, 314–346. BMs represent barriers and filters through which 3 Sanes JR (2003) The basement membranes/basal material diffuses. It is unknown what size and at what lamina of skeletal muscle. J Biol Chem 278, 12601– rate proteins diffuse through BMs. This question is 12604. particularly important for BMs of the vasculature and 4 Kalluri R (2003) Basement membranes: structure, secretory organs, where filtration rate and size exclu- assembly and role in tumor angiogenesis. Nat Rev 6 – sion are key functional elements. Similarly, the mode Cancer , 422 433. 5 Halfter W, Candiello J, Hu H, Zhang P, Schreiber E & of the damage-free passage of neutrophils through Balasubramani M (2013) Protein composition and BMs needs further investigation [101–103]. Studies in biomechanical properties of in vivo-derived basement Caenorhabditis elegans have recently shown that membranes. Cell Adh Migr 7,64–71. anchor cells also pass a BM without leaving a detect- 6 Orkin RW, Gehron P, McGoodwin EB, Martin GR, able breach [104,105]. The passage of the anchor cell Valentine T & Swarm R (1977) A murine tumor was recorded by time lapse, and the recordings indi- producing a matrix of basement membrane. J Exp cated that the intervening BM network is capable of Med 145, 204–220. temporarily expanding, allowing the cell to pass, after 7 Chung AE, Freeman IL & Braginski JE (1977) A which the elastic fibrils of the BM contract and close novel extracellular membrane elaborated by a mouse the temporary opening [104,105]. The fact that cells embryonal carcinoma-derived cell line. Biochem can pass BMs without structural damage infers a fibril- Biophys Res Commun 79, 859–868. lar struture of BMs that is highly flexible and expand- 8 Kleinman H & Martin G (2005) Matrigel: basement able, which is in agreement with the data discussed in membrane matrix with biological activity. Semin this review. Cancer Biol 5, 378–386. Recent data show morphological, compositional and 9 Timpl R, Rohde H, Robey PG, Rennard SI, Foidart functional sidedness of BMs and demonstrate that JM & Martin GR (1979) Laminin–a glycoprotein from BMs are structurally more sophisticated than previ- basement membranes. J Biol Chem 254, 9933–9937. ously considered. The side-specific adhesives function- 10 Chung AE, Jaffe R, Freeman IL, Vergnes JP, ally connects otherwise incompatible epithelia and Braginski JE & Carlin B (1979) Properties of a connective tissues. Taken together, these new findings basement membrane-related glycoprotein synthesized call for a renewed effort aiming to investigate, revise in culture by a mouse embryonal carcinoma-derived 16 – and eventually understand the structual compositions cell line. Cell , 277 287. of in vivo derived BMs and their influence on the 11 Carlin B, Jaffe R, Bender B & Chung AE (1981) Entactin, a novel basal lamina-associated sulfated development and maintenance of epithelia. glycoprotein. J Biol Chem 256, 5209–5214. 12 Timpl R, Dziadek M, Fujiwara S, Nowack H & Wick Acknowledgements G (1983) Nidogen: a new self-aggregating basement membrane protein. Eur J Biochem 137, 455–465. We would like to thank Daphne Asgeirsson for the 13 Hassell JR, Robey PG, Barrach HJ, Wilczek J, critical reading of the manuscript. Rennard SI & Martin GR (1980) Isolation of a heparan sulfate-containing proteoglycan from basement Author contributions membrane. Proc Natl Acad Sci 77, 4494–4498. 14 Kleinman HK, McGarvey ML, Liotta LA, Robey PG, The experimental work was done by Willi Halfter, Tryggvason K & Martin GR (1982) Isolation and Christophe Monnier, Philipp Oertle, Magaly Reyes- characterization of type IV procollagen, laminin, and Lua, Leon Camenzind, Anatalya Labilloy, Manimalha heparan sulfate proteoglycan from the EHS sarcoma. Balasubramani, Haiyu Hu, Joseph Candiello. Willi Biochem 21, 6188–6193. Halfter, Berhard Henrich and Marija Plodinec were 15 Timpl R & Brown JC (1996) Supramolecular assembly laboratory heads and contributed in writing the manu- of basement membranes. Bioassays 18, 123–132. script and designing the experiments. 16 Erickson AC & Couchman JR (2000) Still more complexity in mammalian basement membranes. J Histochem Cytochem 48, 1291–1306. References 17 Miner JH & Yurchenco JR (2004) Laminin functions 20 1 Kefalides NA (1969) The chemistry and structure of in tissue morphogenesis. Ann Rev Cell Dev Biol , – basement membranes. Arthritis Rheum 4, 427–443. 255 284.

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