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Received 1 Sep 2016 | Accepted 9 Oct 2016 | Published 30 Nov 2016

Raphael Reuten1,2,*,w, Trushar R. Patel3,4,*, Matthew McDougall5,*, Nicolas Rama6, Denise Nikodemus1,2, Benjamin Gibert6, Jean-Guy Delcros6, Carina Prein7,8,9, Markus Meier5, Stphanie Metzger10, Zhigang Zhou11,12, Jennifer Kaltenberg1,2, Karen K. McKee13, Tobias Bald14,15, Thomas Tting14,15, Paola Zigrino16, Valentin Djonov17, Wilhelm Bloch18, Hauke Clausen-Schaumann7,9, Ernst Poschl11, Peter D. Yurchenco13, Martin Ehrbar10,Patrick Mehlen6,**, Jrg Stetefeld5,** & Manuel Koch1,2,**

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-afnity binding to the laminin g1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.

1 Institute for Dental Research and Oral Musculoskeletal Biology, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, Cologne 50931, Germany. 2 Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Joseph-Stelzmann-Strasse 52, Cologne 50931, Germany. 3 Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta T1K 3M4, Canada. 4 School of Biosciences, University of Birmingham, Edgbaston B15 2TT, UK. 5 Department of Chemistry, University of Manitoba, Winnipeg R3T 2N2, Canada. 6 Apoptosis, Cancer and Development Laboratory, Equipe labellise La Ligue, LabEx DEVweCAN, Centre de Recherche en Cancrologie de Lyon, INSERM U1052-CNRS UMR5286, Universit de Lyon, Centre Lon Brard, Lyon 69008, France. 7 Center for Applied Tissue Engineering and Regenerative MedicineCANTER, Munich University of Applied Sciences, Lothstrasse 34, Munich D-80335, Germany. 8 Laboratory of Experimental Surgery and Regenerative Medicine ExperiMed, Department of Surgery, Clinical Center University of Munich, Nussbaumstrasse 20, Munich D-80336, Germany.

9 Center for NanoscienceCeNS, Geschwister-Scholl-Platz 1, Munich D-80539, Germany. 10 Laboratory for Cell and Tissue Engineering, Department of Obstetrics, University Hospital Zurich, Schmelzbergstr. 12, Zurich 8091, Switzerland. 11 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK. 12 Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK. 13 Department of Pathology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. 14 Department of Dermatology, University Hospital Magdeburg, Magdeburg 39120, Germany. 15 Laboratory of Experimental Dermatology, Department of Dermatology and Allergy, University of Bonn, Bonn 53105, Germany.

16 Department of Dermatology and Venerology, University of Cologne, Cologne 50931, Germany. 17 Institute of Anatomy, University of Bern, Baltzerstrasse 2, Bern 3000, Switzerland. 18 Institute of Cardiovascular Research and Sport Medicine, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Cologne 50933, Germany. * These authors contributed equally to the work. ** These authors jointly supervised the work. w Present address: Biotech Research and Innovation Centre (BRIC), University of Copenhagen (UCPH), Copenhagen DK-2200, Denmark. Correspondence and requests for materials should be addressed to P.M. (email: mailto:[email protected]

Web End [email protected] ) or to J.S. (email: mailto:[email protected]

Web End [email protected] ) or to M.K.(email: mailto:[email protected]

Web End [email protected] ) or to R.R. (email: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms13515 OPEN

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13515

The netrin protein family is comprised of six members, the secreted netrins -1, -3, -4, -5 and the glycosylphosphatidylinositol (GPI)-anchored netrins-G1 and -G2 in

mammals and exhibit homology to the N-terminal domains (LN-LEa1-3) of laminin short arms18. Secreted netrins-1,-3 and -5 are homologous to the laminin g1 chain, whereas netrin-4 (Net4) shares a higher identity to the b1 chain (50%). Netrin-1 (Net1) has been studied in detail and the name netrin, from the one who guides in Sanskrit was given to this family of laminin-related molecules because of Net1 activity2. Net1 one of the rst studied secreted guidance factors, plays a major role in promoting both axon outgrowth and axon orientation during nervous system development9, as well as in developmental angiogenesis. Given the structural and functional similarities between the axon growth cone and the endothelial tip cell10 in both systems netrin-1 function is due to its ability to engage the receptors DCC and UNC5H (that is, UNC5A, UNC5B, UNC5C and UNC5D)1113 and possibly other receptors such as neogenin, A2b, some integrins or DSCAM1417. Because of the similarity of Net1 and Net4, the majority of studies of Net4 have been focused on describing functions of Net4 based on an assumed interaction of Net4 with DCC or UNC5 and with a guidance function. Works include reported neuronal guidance function3 and pro- as well as anti-angiogenic activity1820. The most prominent in vitro function of Net4 is the inhibition of endothelial tube-like structure formation when Net4 is applied to endothelial cells seeded on Matrigel20. In addition, different cancer cells transfected with Net4 form less-vascularized solid tumours18,19. In most cases, it was speculated that Net4 acts mainly via binding to Net1 receptors of the DCC/neogenin family and UNC5 family members1921. Recently, the binding epitopes of Net1 for DCC, neogenin and UNC5B were identied2224; however, the proposed binding sites are not conserved within Net4. Of interest, even though these studies have received little attention, it was proposed that Net4 binds to the laminin g1 chain25,26.

Together with the fact that most in vitro effects shown with Net4 were obtained from culture in reconstituted matrix such as Matrigel mainly composed of laminin 111, we hypothesized that the Net4 by interacting with laminin g1 could impact on the Matrigel in vitro and on the basement membrane (BM) in vivo.

The BM is a specialized extracellular matrix mainly composed of laminin and collagen IV polymers27 as well as a number of additional proteins. This matrix provides a substrate for cell attachment, serves as a barrier, and is a source of growth factors. As an example, in the vasculature the BM is located at the basal side of the endothelium. The vascular BM (vBM) assembles from proteins produced by endothelial as well as perivascular cells (PVCs)28. Continuous formation of the vBM along the endothelium stabilizes newly formed vessels and promotes their maturation28,29. Ablation of the BM core component collagen IV leads to vascular defects, haemorrhages and embryonic lethality at E10.5 in mouse30. However, these mice still contain BM-like sheets assembled through laminins30. Although, the ultrastructural morphology of BMs is similar between different tissues, variations in protein composition are seen depending on location and during development. Laminins 411 and 511 are the predominant isoforms in the vBM formed by the b1, g1 and either the a4 or the a5 chain. Laminin 411 is expressed in the vasculature during early developmental stages and deciency of the laminin a4 chain leads to a vascular phenotype with transient perinatal hemorrhages31. In contrast, laminin 511 is found in the vBM of adult mice32. Laminin 511, in contrast with laminin 411, contains laminin N-terminal globular (LN) domains on each of its short arms allowing its assembly into a network that is bound to endothelial cell surfaces through integrins, sulfatides and

a-dystroglycan. Following laminin assembly at cell surfaces, collagen IV is integrated into the BM3335.

Here, we present the high-resolution structure of Net4 showing unique features that are absent in Net1 as well as in the N-terminal laminin structures and identify the binding epitopes required for a stable Net4/laminin g1 heterodimeric complex. On the basis of this high-resolution structure, we designed Net4 mutants that negatively impede interaction with laminin g1, possibly explaining previously observed ex vivo effects, on axon outgrowth modulation, and angiogenic activity. Our data also elucidate a mechanism by which Net4 inuence the laminin network and play a vital role in angiogenesis. We also propose that the anti-angiogenic property of Net4 in vivo is directly related to its impact on BM rearrangement.

ResultsNet4 has unique structural features absent in other netrins. In order to establish the structure-function relationship of Net4, we solved the crystal structure of Net4 lacking the C-terminal domain (Net4-DC) at 3.1 (Table 1), revealing a head to stalk arrangement of 175 in length (Fig. 1a,b). The globular shaped N-terminal domain (LN) forms the head and the three and a half rod-like consecutive LE domains make up the stalk (Fig. 1a,b). The LN domain forms a b-sandwich jelly-roll motif with a front face composed of sheet S5-S2-S7 and a dorsal face composed of sheet S4-S3-S6-S1 (Table 1). The topology of this domain is similar to the N-terminal domain of Net1 (refs 2224); however, the closest structural homologue is the short arm of laminin b1 (Supplementary Fig. 1a,b). Signicant structural features make this structure unique among netrins. The LN domain is stabilized by a complex pattern of disulde bridges composed of 12 cysteine residues (Fig. 1a,b). Two disulde bridges between cysteine residues 72236 and 191234, absent in any current netrin or laminin structure, hold the S6-S7 loop in place (Fig. 1b). This loop is adjacent to loop rS1-rS2, which contains the conserved Ser65 (Supplementary Fig. 2a). Interestingly, in laminin b1 (Lmb1), the replacement of this conserved serine by an arginine allows for the formation of a binary complex between b1 and g1 laminins; however, the ternary complex formation with the laminin a chain is abolished. Furthermore, there is clear electron density for glycan additions at three asparagine-linked glycosylation sites, Asn56, Asn163, and Asn353 (Supplementary Fig. 2b). The rst two glycan chains are located at opposite edges of the LN domain, while Asn353 is located at the loop b helix of LE2 and extends towards the bottom of the LN domain (Fig. 1a,b). Remarkably, Net4 is the rst member of the netrin family with no N-linked glycan attachment at the dorsal face of the N-terminal domain. Net4-DC has a total accessible surface area of B28,000 2 and reveals extensive inter-domain contacts between the LN domain and subdomain LE1 (Fig. 1a,b). Loop b of the LE1 domain has a large buried surface area of B1,700 2 with the lateral edge of the LN domain (Fig. 1c). In addition, loop d of the LE1 domain is anked by the Asn353-linked glycan attachment and trapped between the N-terminal segment and the basal helix of the LN domain (Fig. 1d). Together, these extensive contact areas may limit the rotational freedom of the LN domain in comparison with other netrins. Each of the individual LE-subdomains adopts the classical LE-fold36 consisting of irregular coil segments and forms a linear extended structure.

Net4 does not interact with Net1 receptors. As the DCC and UNC5B-binding epitopes of Net1 are absent in the Net4 structure2224, we examined whether Net4 could interact with the ectodomains of DCC, UNC5B, A, C, D and more generally A2b, neogenin as well as DSCAM that have been previously reported

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13515 ARTICLE

a b c

N163 N56

Ca2+

rS1

rS2

Domain LE1

Domain LE3

Domain LE2

Domain LE31/2

Domain LN

Ca2+

H2 S4

loop b of LE1

S9 H4

N353 LE1

S7 Ca

S11

S10

LE2

LE3

Net1

loop d of LE1

N353

e f

Net4

DCC DCC

F441

M933 M933

N370

N360

R348 R349

D339

T340

L446 R372

Q363

Net1

Net4

DCC DCC

g h

Net1

Net4

M959 M959 F441 L446

Neogenin Neogenin Neogenin

Net1

Figure 1 | Overall structure of Net4. (a) Ribbon model of Net4-DC viewed in the open face orientation. The domains LN (teal), LE1 (orange) LE2 (magenta) and LE3-31/2 (green) are coloured accordingly. N-linked glycans are drawn as red sticks, disulde bridges are drawn as yellow sticks, and the calcium ion as a red sphere. (b) Secondary structure diagrams. Disulde connectivity in domains LN and LE are indicated by yellow dotted lines, visible N-linked glycans by green stars with the Asn residues N56, N163 and N353 highlighted. b-strands are depicted as arrows and the a-helices as cylinders, each labelled with the rst and last residue. The individual loop segments are shown in different colour codes. (c) Interface between domains LE1 and LN. The

KAPG motif is free in solution and not visible in the structure. (d) Secondary interface between domains LE1 and LN. The loop d segment between Cys307 and Cys329 of subdomain LE1 is in contact with the N-terminus and the loop S5-H3 of domain LN. (e) Complex of Net1 (marine; PDB 4OVE) and DCC (orange; PDB 4URT) at site 1 with interacting residues shown at full opacity and the same complex with Net4 (green) replacing Net1. (f) Complex of Net1 (marine) and DCC (orange; PDB 4URT) at site 2 with interacting residues shown at full opacity and the same complex with Net4 (green) replacing Net. (g) Complex of Net1 (marine) and neogenin (pink; PDB 4PLN) at site 1 with interacting residues shown at full opacity and the same complex with Net4 (green) replacing Net1. Similar to site 1 of the DCC-Net1 complex, F441 (red) of Net4 occludes Met959 of neogenin from the hydrophobic pocket. L456 (red) of Net4 again clashes with the binding partner. (h) Net1 (marine) in complex with neogenin (pink) at site 2. Note the difference in the surface charge at the neogenin binding site (indicated by a box) of Net1 (left) and the corresponding site on Net4 (right).

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13515

Table 1 | Data collection and renement statistics.

Net4-DC

Data collectionSpace group C121Cell dimensionsa, b, c () 107.755, 74.049, 74.672 a,b,g () 90, 96.072, 90

Resolution () 46.00-3.07 (3.12-3.07)* No. reections 39327 (1952) Rmerge 0.194 (0.776)

I/sI 6.10 (2.0) Completeness (%) 99.9 (99.8)

Redundancy 3.5 (3.5)

RenementRwork/Rfree 0.2198/0.2626

No. atoms 3,382

Protein 3,259 Ligand/ion 96 Water 27 B-factors 45.90

Protein 44.10 Ligand/ion 110.30 Water 32.60 R.m.s. deviations

Bond lengths () 0.003 Bond angles () 0.77

Ramachandran StatisticsFavoured (%) 92.2 Disallowed (%) 0.0

*Values in parentheses are for highest-resolution shell.

to interact with Net1, using solid-phase binding assays and Octet technology. Structural analysis of Net4 and previously published Net1 complex structures reveals incompatibility between the binding residues of Net1 and equivalently positioned residues of Net4 (Fig. 1eh). For example, Phe441 of Net4 occludes the conserved Met from the hydrophobic pocket in both DCC and neogenin (Fig. 1e,g), and Leu458 of loop b in the terminal LE domain of Net4 would preclude interaction with the netrin receptor. The absence of the positively charged residues of Net1 (Arg348, Arg349, Arg351 and Arg372) necessary for a sulfate-mediated interaction with DCC in Net4 would hinder interaction between DCC and Net4 (Fig. 1f). The binding of neogenin to the LN domain involves 10 residues of Net1, which are completely non-conserved in Net4, as presented by the electrostatic surface potential (Fig. 1h). The buried surface area of this interaction is B680 2, and covers a large portion of the LN domain of Net1.

None of the residues from Net1 that interact with neogenin are conserved in Net4 (Fig. 1h).

While Net1 interacts in the nanomolar range with DCC and UNC5B, Net4 does not show any interaction with any of the proposed Net1 receptors including DCC/neogenin, UNC5 and integrins (Fig. 2a and Supplementary Fig. 3). Sequence alignments of netrin family proteins further highlight that almost all residues involved in Net1 interaction with the canonical netrin receptors are not conserved in Net4 (Supplementary Fig. 4). Together these observations support the view that Net4 does not interact at least directly with the known Net1 receptors, even though we can discard that in some specic settings a protein complex may include Net4 and Net1 receptors.

Net4 forms a high-afnity complex with laminin c1. Although full-length Net4 is reported to exist as a dimer3 and is therefore believed to bridge two laminin heterotrimers, our crosslinking

experiments as well as hydrodynamic studies reveal that full-length Net4 (Net4-FL) forms a high-afnity complex with laminin g1 (Lmg1) in a 1:1 molar ratio (Supplementary

Fig. 5ae). The hydrodynamic radius (rH) from dynamic light scattering (5.770.02 nm) and the sedimentation coefcient from analytical ultracentrifugation (AUC, 5.150.01 S) conrm unambiguously the existence of an equimolar heterodimeric complex with a molecular mass of 116 kDa. The rotary shadowing experiments reveal a central globular structure with two elongated rods oriented in opposite directions, consistent with a LN domain-mediated major binding interface with both elongated LE subdomains oriented in an antiparallel fashion (Fig. 2c). Small angle X-ray scattering (SAXS) studies of g1LN-LEa1-4 in complex with full-length Net4 (Net4-FL) yield an extended conformation with a maximum particle size (Dmax) of 19 nm (Supplementary Fig. 5d and Supplementary Table 1). Rigid body tting allows the reconstruction of the assembly of full-length Net4 and laminin g1 (Net4-FL-g1LN-LEa1-4) in contact through the LN domains, with the C-terminal domain of Net4 (domain C) curling back over the LE domains. As presented in Fig. 2b, both N-terminal b-sandwiches are oriented like the palms of two hands with the open face sheets S5-S2-S7 (Net4) and S6-S3-S8 (laminin g1) in contact with each other (Fig. 2b and

Supplementary Movie 1). The models were validated extensively by comparing experimentally determined hydrodynamic properties with model derived hydrodynamic properties as presented in the Supplementary Table 1. The comparison of Net4-DC and Net4-FL binding to laminin g1 indicated equal afnities in the range of KD 2 nM, validating the exclusion of

domain C of Net4 in complex formation (Fig. 2d). To decipher individual amino acid residues responsible for Net4-laminin g1 interactions, we designed mutants based on the three-dimensional structure of Net4 as well as the phylogenetic conservation (Supplementary Fig. 1a,b). A structure-guided deletion of the unique Net4 loop b (KAPGA) resulted in a 25-fold reduction of KD from 2 nM (Net4-DC) to B50 nM (Net4-DCDKAPGA) (Supplementary Fig. 6a,b). Thus, Net4-DCDKAPGA emphasizes the importance of the KAPGA loop in complex formation and gives a rationale for the lower afnity of laminin b1 to laminin g1 (KD of 5 mM)33,35, as this loop is only present in Net4. Moreover, we identied two residues (E195 and R199) within the LN domain of Net4 involved in laminin g1 binding. Mutation of these two residues results in a decreased KD from 2 to 66 nM (Supplementary Fig. 6c). In addition, mutation of E195 as well as R199 together with deletion of loop b (Net4-DCE195A,R199A,DKAPGA) completely abolishes binding in all performed binding assays (Fig. 2d and Supplementary Fig. 6a,b). Crosslinking experiments conrm that full-length Net4 (Net4-FL) exists as a monomer and dimer, because increasing crosslinker concentration results in an increase in dimeric and multimeric Net4-FL (Supplementary Fig. 5a). The rH of Net4-FL was determined to be 5.760.02 nm which is very similar to that of Net4-FL-g1LN-LEa1-4 (5.770.02 nm) and Net4-DC-g1LN

LEa1-4 (5.400.10 nm) complexes but signicantly higher than Net4-DC (4.600.20 nm), implying that full-length Net4 (Net4-FL) behaves as a mixture of monomer and dimer in solution (Supplementary Fig. 5e and Supplementary Table 1). The maximum particle size (Dmax) calculated from SAXS data for

Net4-FL is signicantly higher than the Dmax of Net4-DC, but similar to both complexes (Supplementary Fig. 5e and Supplementary Table 1). The AUC studies of Net4-FL provided two peaks corresponding to a monomer and to a dimer for Net4-FL, while Net4-DC (ref. 37) and complexes of Net4-FL and

Net4-DC with g1LN-LEa1-4 yield only a single peak (Supplementary Fig. 5e). These experiments provide evidence that full-length Net4 (Net4-FL) exists as a mixture of monomer

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13515 ARTICLE

DCC Neogenin UNC5B

Net1 Net4

Binding (nm)

0.2

0.1

0.0

0.4

0.2

Binding (nm)

0.2

0.1

0.0

200

400 Time (s)

Domain C

600 200 400

Time (s)

600

200 400 Time (s)

600

1LN-LEa1-4

LEa1 LEa3

LEa2

LE2

LEa4

LE31/2

LE1

LE3

Net4-FL

Net4-C Net4-CE195A,R199A,KAPGA

Net4-FL

700

F norm(1/1,000)

700

680

660 101 100 101

Analyte (nM)

700

680

KD: 2.29 0.53 nM KD: 2.26 0.70 nM

680

660 101 100 101

Analyte (nM)

F norm(1/1,000)

n.b.

102 103 104

660 101 100 101

Analyte (nM)

102 103 104

e f g

R199

E195 mNet4 181 203

208 231 204

296 308 278

mNet1 mNet3

mNet4 mNet1 mNet3

177

269 292 262

Lm111

L217

Fraction bound

S221

1.0

Net4

Net1

0.5

0.0

100 101 102 103Analyte (nM)

Figure 2 | Binding comparison of Net4 and Net1 to netrin receptors and laminin. (a) Biolayer interferometry binding kinetic analysis of DCC, neogenin, and UNC5B to Net4 (red) as well as Net1 (green). AHC sensors were coated with the Fc chimera proteins. Association (from 0 to 300 s) and dissociation (from 300 to 600 s) phases are shown for 50 nM of both netrins. (b) The SAXS model for the Net4-FL-g1LN-LEa1-4 complex was generated by rstly calculating models for Net4-DC-g1LN-LEa1-4 complex (Supplementary Fig. 5d) followed by the addition of the LE and globular domain C. This model highlights the role of N-terminal globular domains in the interaction. The agreement between experimentally collected SAXS data and model-derived SAXS data is presented in Supplementary Table 1. (c) Rotary-shadowing electron microscopy image of Net4-DC-g1LN-LEa1-4 clearly showing a 1:1 complex mediated via the globular domains. (d) Microscale thermophoresis binding analysis of different Net4 mutants to labelled g1LN-LEa1-4. Binding of Net4 lacking the netrin-specic C domain (Net4-DC). Binding of full-length Net4 (Net4-FL). Analysis of the binding of a Net4 mutant (Net4-DCDKAPGA) in which the specic loop b (KAPGA) within LE1 was deleted. Binding of a combined Net4 mutant (Net4-DCE195A,R199A,DKAPGA). Error bars, s.d. (n 3 independent

technical replicates). KD values are shown in the graph (n.b., no binding). (e,f) Laminin-binding epitopes within Net4 (green) compared with the equivalent positions in Net1 (marine). Note loop b of LE1 of Net4 has an extensive contact area with the LN domain, and the KAPGA motif (shown in stick notation) is highly exible. (g) Sequence alignment of Net4, Net1 and netrin-3 showing the laminin-binding region. (h) Solid-phase binding study of Net4 and Net1 to immobilized laminin g1 (g1LN-LEa1-4). Error bars, s.d. (n 3 independent technical replicates). Scale bar, (c) 100 nm.

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13515

and dimer in solution and that laminin g1 is capable of destabilizing the Net4-FL dimer. Taken together, our results establish that full-length Net4 binds to laminin g1 in a 1:1 stoichiometry. In addition, biochemical studies identied different epitopes within Net4 responsible for laminin interaction. Our newly established Net4 laminin-binding mutants can now be used to determine the specic role of the Net4-laminin complex in functional studies because these mutants do not affect cell adhesion properties of Net4 (Supplementary Fig. 6d) indicating their exclusive role in laminin binding. Moreover, comparing the structure of Net4 and Net1 reveals that the laminin-binding epitopes are not conserved within Net1 (Fig. 2eg) and indeed Net1 does not interact with laminin 111 (Fig. 2h).

Net4 disrupts pre-existing laminin networks. Net4 has been proposed to block laminin polymerization25. However, due to its nanomolar binding to the laminin g1 chain, we hypothesized that

Net4 not only blocks laminin polymerization but also disrupts pre-existing laminin network. First, we analysed whether Net4 and the identied laminin-binding mutant (E195A,R199A), which still binds in a nanomolar range to the laminin g1 chain, are able to block laminin polymerization (Fig. 3a and Supplementary Fig. 7a). Interestingly, Net4-DC as well as the laminin-binding mutant Net4-DCE195A,R199A blocked laminin polymerization to the same extent as the N-terminal fragments of laminin b1 and g1 (Fig. 3b). To address our hypothesis that Net4 is able to disrupt pre-existing laminin networks, we performed a comprehensive series of different techniques, including(i) laminin network disruption (Supplementary Fig. 7b),(ii) size-exclusion chromatography and (iii) atomic force microscopy of a polymerized Matrigel matrix. Net4 is able to disrupt pre-existing laminin 111 networks and the ternary node complex formed by the laminin a1, b1 and g1 chain (Fig. 3ce).

Moreover, Net4 treatment dramatically weakens the stiffness of the Matrigel matrix (Fig. 3f). Thus, all approaches clearly highlight that Net4 is able to disrupt pre-existing laminin networks (Fig. 3cf). However, the laminin-binding mutant Net4-DCE195A,R199A is only able to block laminin polymerization but not to disrupt pre-existing laminin networks (Fig. 3c,d,f). Our results clearly demonstrate that N-terminal laminin fragments and Net4, as proposed previously25,33 as well as the laminin-binding mutant, are able to block laminin polymerization due to their binding capacity to the N-terminal laminin domain (LN) (Fig. 3g). These results establish that Net4 is able to disrupt pre-existing laminin networks in a non-enzymatic manner, whereas its respective laminin-binding mutant cannot (Fig. 3g).

Net4 interferes with BM assembly on the cell surface. As Net4 has a high-afnity interaction with laminin g1, a key component of the BM, we investigated whether Net4 affects BM assembly. As an in vitro model, we rst took advantage that BM components assemble on Schwann cell surfaces only if laminin 111 heterotrimers interact via their LN domains34. Therefore, the stable heterodimeric complex of Net4 with laminin g1 may prevent efcient BM assembly on Schwann cell surfaces similar to LN domain truncated laminin 111. Schwann cells were incubated with laminin 111, collagen IV and nidogen-1, which normally results in a BM assembly on the cell surface34. Deposition of BM components was then monitored via immunouorescence staining for collagen IV (Col-IV) and laminin (Lmg1) (Fig. 4a).

First, laminin 111 binds to cell surface integrins via its C-terminal LG domains and assembles via the LN domains of the a1, b1 and g1 chain. Both interactions are necessary for laminin deposition34. We observed strong laminin as well as collagen IV staining when treating cells with only laminin 111, collagen IV

and nidogen-1 (ctrl) (Fig. 4a,b). After addition of wild-type Net4 (Net4-FL) together with BM components, we could neither detect laminin nor collagen IV deposition by immunouorescence, whereas treatment with Net4-FLE195A,R199A showed strong laminin as well as collagen IV staining as observed in the control situation (Fig. 4a,b). Moreover, we analysed the inuence of different molar ratios of laminin 111 to Net4-FL as well as Net4-FLE195A,R199A (1:0.4, 1:1.8, 1:7 and 1:28). Net4 signicantly decreased laminin as well as collagen IV levels on the cell surface at ratios 1:7 and 1:28, whereas the laminin-binding mutant (Net4-FLE195A,R199A) had no inuence on laminin and collagen IV staining intensities (Fig. 4c). In summary, these results indicate that Net4 can destabilize the polymerization of laminins containing the laminin g1 chain followed by disassembly of the BM.

Net4 may affect axon outgrowth through matrix reorganization. Net4 has been postulated to play similar roles as Net1, mainly due to their conserved domain features3840. Furthermore, it has been proposed that Net4 acts as an outgrowth promoting cue in ex vivo models such as the olfactory bulb neurite outgrowth3. We revisited this axon outgrowth function in the light of Net4s impact on the extracellular matrix. Olfactory bulb explants from E15 embryos were cultured in collagen I in the presence of the N-terminal laminin g1 fragment, Net4-DC, Net4-DC together with the N-terminal laminin g1 fragment and the laminin-binding mutant Net4-DCE195A,R199A, which does not interact with laminin (Fig. 5a). As shown in Fig. 5b, while wild-type Net4 presence is associated with neurite outgrowth, the combination of Net4 with the N-terminal laminin g1 fragment as well as the laminin-binding mutant was unable to promote axon outgrowth (Fig. 5b,c). These data support the view that, unlike Net1, the axon outgrowth activity of Net4 is not only due to guidance cue functionality per se. It may additionally be due to Net4 ability to disrupt the BM surrounding the olfactory bulb through interactions with laminin-supporting axons to branch through this physical barrier, even though we cannot exclude other interpretations. To further analyse this indirect function, we next focused on the described role of Net4 on angiogenesis.

Net4 affects angiogenesis through matrix reorganization. Most studies investigating the role of Net4 in angiogenesis in vitro or ex vivo are using articial matrix for the culture in vitro19,20,4144. The role of a functional laminin network within articial matrix such as Matrigel for the formation of tube-like structures has so far not been addressed. A recent study revealed that Net4 inhibits the formation of endothelial tube-like structure at high concentrations (1,000 nM) (ref. 20). To conrm this, Human dermal microvascular endothelial cells (HDMECs) were seeded on Matrigel and different concentrations (0, 200 and 1,000 nM) of Net4 (Net4-DC) were added after cell attachment on Matrigel (Supplementary Fig. 8a). Untreated HDMECs formed cell clusters connected by honeycomb-like network structures after 14 h. Upon addition of 200 nM Net4 (Net4-DC), endothelial cells were able to form networks, whereas treatment with 1,000 nM Net4 completely abolished tube-like structure formation (Fig. 6a and Supplementary Fig. 8a,b). In addition, HDMEC tube-like structure formation was monitored for 14 h (Supplementary Movies 2 and 3); upon Net4 treatment (1,000 nM) cell morphology was dramatically changed and no network was established. Instead, cells moved together and formed pronounced aggregates within 5 h and remained clustered over the 14 h period (Supplementary Fig. 8c). On the basis of our hypothesis that Net4 disrupts laminin-laminin complexes, we further investigated whether Net4 alters tube-like structure

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formation in a cell-type independent manner. Upon Net4 treatment, tube-like structure formation in microvascular (HDMEC), macrovascular (HUVEC) and lymphatic (HDLEC) endothelial cell lines was inhibited compared with control (Supplementary Fig. 8d). In addition, Net4 also destabilizes an

already established vessel-like network on Matrigel 7 h post treatment followed by increased cell aggregation after 14 h (Supplementary Fig. 8e). In a subsequent step, we further determined whether the exchange of the LN-LEa1 domains within the laminin g1 chain fragment with the respective

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domains (LN-LEa1-4) of the laminin g1 chain (Lm111Dg1LNLEa1-4)34. As expected, Net4 does not bind to Lm111Dg1LNLEa1-4 (Supplementary Fig. 11a), whereas Agrin-Net4-DC and the laminin-binding mutant fused to the N-terminal agrin domain (Agrin-Net4-DCE195A,R199A) interact in the nanomolar range with

Lm111Dg1LN-LEa1-4 (Supplementary Fig. 11b). Treatment of HDMECs with Agrin-Net4-DC abolished tube-like structures to the same extend as wild-type Net4 (Net4-DC and Net4-FL, Supplementary Figs 9a and 11c). However, the laminin-binding mutant fused to the N-terminal agrin domain as well as the combination of Agrin-Net4-DC together with the laminin g1LN

LEa1-4 fragment signicantly blocked the activity of Net4 (Supplementary Fig. 11c). Thus, our data support the view that a formed laminin network within the Matrigel matrix is essential to generate tube-like structures and that the reported inhibitory activity of Net4 is mediated through binding to the LN domain of the laminin g1 chain and disruption of the BM.

To unravel the physiological signicance of our ndings we performed a more physiological relevant angiogenesis assay, where endothelial cells were co-cultured with pericytes to form tubes within a laminin-free collagen I matrix28. Here, endothelial cells and pericytes form a continuous BM formed by laminin 511 and collagen IV. Treatment of the co-culture with Net4 completely abolished tube formation, whereas the laminin-binding mutant showed no effect (Fig. 6d). Staining of endothelial tubes highlight that laminin as well as collagen IV is not present along the tubes in the Net4 treated set up (Fig. 6e).These results not only suggest that Net4 disrupts the laminin network resulting in the decline of endothelial tubes but also emphasize that the laminin network is the key component of the vBM essential for vessel formation.

The Net4/laminin interaction destabilizes capillaries in vivo.

To determine how Net4 affects capillary networks an ex ovo chicken Chick Chorioallantoic Membrane (CAM) angiogenesis assay46 was performed. Established capillary networks were treated with full-length Net4 (Net4-FL) and the laminin-binding mutant (Net4-FLE195A,R199A); blood and micro-capillaries were visualized using FITC-dextran injection 48 h post treatment. In the untreated (ctrl) CAMs, a micro-vascular network appeared 48 h post treatment. Upon addition of Net4, the capillary network dramatically changed as the observed vascularized area was signicantly decreased (Fig. 7a). Treating the CAMs with the laminin-binding mutant Net4-FLE195A,R199A had no signicant effect. Histological staining (haematoxylin, eosin; H&E) of CAMs to detect the capillary density of untreated

Figure 3 | Net4 disrupts pre-existing laminin networks. (a) Inhibition of laminin 111 polymerization (Lm111) by human laminin b1 (b1LN-LEa1-4), mouse laminin g1 (g1LN-LEa1-4), mouse Net1 (Net1-DC), mouse Net4-DC and the laminin-binding mutant mouse Net4-DCE195A,R199A. For the experimental scheme depicting the laminin 111 polymerization assay see Supplementary Fig. 7a. SDSPAGE analysis of the pellet (P) and the supernatant fraction (S). The position of the laminin chains (arrow heads) and the recombinant proteins (stars) are indicated. (b) Densitometric analysis of three independent polymerization experiments and the percentage of the pellet fraction are displayed. (means.d.; n 3; ****Po0.00006 (ctrl vs b1LN-LEa1-4, Net4-DC and

Net4-DCE195A,R199A) and ***P 0.0004 (ctrl vs g1LN-LEa1-4); ns, not signicant). (c) For the method procedure see Supplementary Fig. 7b. The resulting

fractions (supernatant of laminin polymerization (S), supernatant after addition of different proteins (S ) and polymer (P )) were separated by

SDSPAGE, followed by Coomassie Brilliant-Blue staining. Different laminin chains are colour labelled (a1 (yellow), b1 (green) and g1 (blue)) and added recombinant proteins are indicated with asterisks. (d) The ratio between polymer (P ) and supernatant (S ) was determined by densitometric analysis

of at least three independent experiments (means.d.; n 3; ****P 0.00005). (e) N-terminal laminin fragments (LN-LEa1-4) of a1 (yellow), b1 (green)

and g1 (blue) as well as MBP-Net4-DC (red) single proteins were analysed using analytical size-exclusion chromatography (top). SEC prole of the laminin fragment g1LN-LEa1-4 (g1) in complex with MBP-Net4-DC (green, middle). Complexes [a1-b1-g1 (blue) and a1-b1-g1 MBP-Net4-DC (red)] were analysed

by SEC (bottom) revealing that the MBP-Net4-DC is able to disrupt the a1-b1-g1 complex. Asterisks indicate the single proteins within the complex. (f) Atomic force microscopy anaylsis of a polymerized Matrigel matrix treated with Net4-DC or with the laminin-binding mutant Net4-DCE195A,R199A.

(g) Model depicting inhibition of laminin polymerization via laminin b1 (b1LN-LEa1-4), laminin g1 (g1LN-LEa1-4), mouse Net4-DC, and the laminin-binding mutant mouse Net4-DCE195A,R199A but not via mouse Net1 (Net1-DC). The pre-existing laminin network can only be solubilized through Net4. Error bars, s.d. (n 3 independent technical replicates).

laminin-binding domains of Net4 LN-LE1 might lead to gain of function. Both chimeric fragments (chimera 2: Net4LN-LE1-2g1LEa3-4 and chimera 3: Net4LN-LE1g1LEa2-4) inhibited endothelial tube-like structure formation in the same way as Net4-DC, whereas the chimeric fragment (chimera 1: g1LN

LEa1Net4LE2-31/2) did not show any alteration of endothelial tube-like networks at the concentrations tested (Supplementary Fig. 8f).

To determine whether alterations in the laminin network are the trigger of Net4 mediated inhibition of tube-like structures, we performed the Matrigel assay with the structure-guided Net4 laminin-binding mutants (Net4-DCE195A, Net4-DCR199A, and

Net4-DCDKAPGA and Net4-DCE195A,R199A). HDMECs treated with these laminin g1 binding mutants established cell clusters with lamentous interconnections in the same manner as untreated and laminin g1LN-LEa1-4 fragment treated endothelial cells (Fig. 6a, left; Supplementary Fig. 9a,b). Net4 signicantly decreased cell cluster interconnections (tube number), whereas tube numbers in g1LN-LEa1-4- and Net4 laminin-binding mutants-treated endothelial cells were equal to untreated endothelial cells (Fig. 6a, right; Supplementary Fig. 9a,b). The inhibition of tube-like structure formation through Net4 was rescued via pre-incubation of Net4 together with the laminin-binding domain of the laminin g1 chain (g1LN-LEa1-4). Analysis of endothelial tube numbers showed no difference between untreated, treatment with g1LN-LEa1-4, and combined treatment of g1LN-LEa1-4 together with Net4 (Fig. 6b and Supplementary Fig. 9a,b). This nding demonstrates that the laminin g1 fragment competes with the interaction of Net4 to the laminin g1 chain of the laminin 111 heterotrimer. To conrm the cell-independent activity of Net4, we also investigated the inuence of Net4 on the tube-like structures45 made by the mouse melanoma B16-F1 cell line. Here, Net4 could also block the formation of tube-like structures in a laminin-binding-dependent manner (Supplementary Fig. 10a,b). Furthermore, we analyzed the effect of netrin-4 on endothelial cells in a laminin-free matrix is in agreement with the presented model: addition of netrin-4 had no effect on endothelial cell sprouting in a collagen I matrix (Fig. 6c). To exclude that the observed activity of Net4 is only mediated when Net4 is deposited within the laminin network of the Matrigel matrix, we designed a Net4 protein fused to the N-terminal agrin domain (Agrin-Net4-DC). First, we tested the binding activity of Agrin-Net4-DC to the laminin g1LN-LEa1-4 fragment. This construct binds with the same afnity to the laminin g1LN-LEa1-4 fragment as wild-type Net4 (Supplementary

Fig. 6c). Moreover, we tested the binding activity of the N-terminal agrin domain to laminin 111. Here, we took advantage of a previously generated laminin 111 construct lacking the N-terminal

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Figure 4 | Net4 blocks laminin assembly on the cell surface. (a) Within untreated (ctrl), Net4-FL-, and Net4-FLE195A,R199A-treated (28-fold excess in

molarity compared with laminin) Schwann cell extracellular matrix assembly assay BM components collagen IV (Col-IV, green) and laminin 111 (Lmg1, red) were stained. Cells were counter-stained for DAPI (blue). (b) Staining intensities are displayed relative to the DAPI signal [relative Lmg1 intensity per cell (left) and relative Col-IV intensity per cell, (right)] (means.d.; n 5; ****Po0.00001; ns, not signicant). Error bars, s.d. (n 5 independent cell

cultures). (c) Concentration dependent effect of Net4-FL and Net4-FLE195A,R199A on BM formation indicated through measurement of the relative Lmg1 intensity/cell (left) and relative Col-IV intensity/cell (right). Net4 proteins were added together with the BM components laminin 111, collagen IV and nidogen-1 with increasing molar ratios to laminin 111 (0.4, 1.8, 7, 28-fold excess). Scale bars, (a) 200 mm. P values were calculated by one-way ANOVA followed by a pairwise comparison using the Holm-Sidak method. ANOVA, analysis of variance.

and Net4-FLE195A,R199A-treated CAMs displayed a dense capillary lining, whereas Net4-treated CAMs revealed fewer and more rounded capillaries (Fig. 7b).

One of the key pathophysiological roles of Net4 associated with its angiogenic activity is its inhibiting effect on tumour progression. Subcutaneously injected cancer cells overexpressing Net4 in mice establish tumours with fewer vessels than tumours established by untransfected cells18,19. Therefore, we investigated whether the tumour-suppressive activity of Net4 is mediated through binding to laminin. HCMel12 mouse melanoma cells were injected in mice and, upon macroscopic detection; tumour growth was monitored and daily injection of either a control protein (mouse serum albumin), Net4 or its laminin-binding mutant was administered (Fig. 7c). The size of tumours treated with Net4 was reduced by 50% of the size in comparison to the control-treated animals. Moreover, our data establish that the interaction of Net4 with laminin is essential for its tumoursuppressive activity as the Net4 mutant (Net4-FLE195A,R199A), affected in laminin binding, had no impact on tumour growth (Fig. 7d,e).

We thus moved back to the ex ovo CAM model to further dissect the impact of Net4 on capillaries. We investigated in CAMs treated with Net4 or its laminin-binding mutant, the branching pattern that is highly altered only in the Net4-treated CAM. To highlight the capillary branching hierarchy, the vessel diameters were presented in a radial graph. This analysis clearly indicates that diameters of vessels within Net4-FLE195A,R199A-treated CAMs span the full range of diameters found in an

established vessel hierarchy. In contrast, vessels in Net4-FL-treated CAMs showed an altered distribution of diameters as large vessels exhibited smaller diameters and small capillaries were not present (Fig. 8a, left). Net4-FLE195A,R199A-treated CAMs display a distinct established hierarchy pattern, starting with large vessels (1., 4560 mm diameter) from which smaller capillaries branch with decreasing diameter (2., 3045 mm; 3., 1530 mm; and4., 36 mm). The altered vessel hierarchy in Net4-treated CAMs is clearly demonstrated by decreased diameters as large vessels(1., 2837 mm) as well as small capillaries (2., 1020 mm and 3., 710 mm) exhibit reduced calibers (Fig. 8a, right). Thus, the lamininlaminin interaction is required to maintain proper capillary networks in vivo.

As Net4 appears to disconnect the laminin network ex ovo, we hypothesized that the disruption of the laminin network may affect the whole vBM thereby inuencing the interaction of pericytes with the endothelium. Therefore, endothelial and perivascular cell distribution was studied in CAMs treated with the laminin-binding mutant (Net4-FLE195A,R199A) or Net4. Ultrastructural analyses of Net4-FLE195A,R199A-treated CAMs suggested that endothelial cells are closely surrounded by pericytes, whereas Net4 treatment leads to detachment of pericytes from the endothelium with altered pericyte morphology (Fig. 8b, top). Embedding of pericytes into the vBM stabilizes newly-formed vessels. Net4-FLE195A,R199A-treated CAMs exhibited a continuous vBM along the endothelium and surrounding pericytes. Upon Net4 treatment, the vBM almost disappeared on endothelial cell surfaces as well as along pericytes (Fig. 8b, middle

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Figure 5 | Net4 induces neurite outgrowth in a laminin-dependent manner. (a) Diagram depicting the olfactory bulb (OB) explants assay procedure. (b) Representative images of untreated (ctrl), laminin g1 (g1LN-LEa1-4)-, Net4 (Net4-DC)-, Net4 in combination with laminin g1 (Net4-DC g1LN-LEa1-4)-

, and Net4 laminin-binding mutant (Net4-DCE195A,R199A)-treated OB explants. (c) Statistical analysis of OB explants (means.e.m.; n 19; ****Po0.0001,

ns, not signicant). Error bars, s.e.m. (n 19 independent cell cultures). P values, two sided t-test. Scale bar, 200 mm (b).

and bottom). These results indicate that Net4 dissociates laminin heterotrimers from the vBM and disturbs the endothelial/ perivascular cell interaction in vivo.

DiscussionFar from its expected role as a guidance cue, a combination of structural, in vitro and in vivo experiments demonstrate that Net4 behaves in a completely different manner than its well-studied family member Net1. Our data demonstrate that Net4 not only strongly binds to laminin g1 -thereby blocks efciently laminin polymerization- but also actively disrupts pre-existing laminin networks in a non-enzymatic manner. Net4 is clearly a unique member among the netrin family, showing non-enzymatic disruptive forces towards matured BMs.

It has been reported that Net4 demonstrates axonal growth activity3 and anti-angiogenic activity in vitro by inhibiting endothelial tube-like structure formation1820. Furthermore, Net4 overexpression in different cancer cells reveals a decreased vascularization in vivo18,19. Even though we cannot discard that Net4 is having an alternative function via unknown receptor, we propose here that the basis of the effects reported on axonal growth, angiogenesis and tumour progression is due to disruption of pre-existing laminin networks resulting in the disruption of the entire BM. Endothelial assemblies need to form a continuous vBM together with pericytes28. Our treatment studies with Net4 conrm this fact due to the fact that the disruption of the vBM results in the decline of endothelial tubes. In terms of the effect of Net4 on neurite outgrowth in the olfactory bulb assay the role of

the BM is different. Here, axons need to branch through the BM as a physical barrier and the disruption of this barrier through Net4 supports axonal outgrowth. Interestingly, most of the cellular effects reported on Net4 have been performed using an articial matrix such as Matrigel that contains mainly laminin 111 (refs 19,20,47,48). As the LN domain of the g1 chain is the major binding partner of Net4, we hypothesized that it affects the laminin network within the Matrigel matrix instead of directly affecting cell-signalling pathways. Similarly inhibition of blood vessel formation by Net4 is believed to be triggered through binding to the Net1 receptors DCC (DCC, neogenin) and UNC5 (UNC5A-D)18,19. However, we could not detect direct interactions of Net4 with any of these receptors. Comparison of the high-resolution structures of Net1 (refs 2224) and Net4 explains in detail this absence of interaction (Fig. 1eh and Supplementary Fig. 1a).

Structure based mutations of Net4 in combination with in vivo and ex vivo assays allowed assignment of Net4 functionality to its laminin-binding capability rather than Net4s interaction with Net1 receptors. Thus, our hypothesis is that Net4 is not a classic ligand activating a membrane receptor with a downstream signalling but rather a regulator of basal membrane maintenance, which indirectly impacts key functions such as axonal growth and angiogenesis. Our study rather support the view that Net4 is a unique protein able to disrupt laminin g1 containing BMs and this can turn as an useful tool be used to investigate the pivotal role of lamininlaminin interactions in biological processes, for example, muscle formation, somitogenesis and neural tube formation.

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Figure 6 | Net4 activity on angiogenesis is dependent on Net4/laminin interaction. (a) Analyses of tube-like structure inhibition by Net4-DC mutants (Net4-DCE195A, Net4-DCR199A and Net4-DCDKAPGA) impaired in laminin g1 binding (protein concentrations: 1 mM). Statistical analyses of tube formation via determining the number of cell cluster connections (tube number) (means.d.; n 3; ***P 0.0007) (right). Error bars, s.d. (n 3 independent cell cultures).

(b) Blocking of Net4-DC inhibited tube formation through equimolar addition of g1LN-LEa1-4 (1 mM, molar ratio of Net4-DC:g1LN-LEa1-4, 1:1). The laminin fragment g1LN-LEa1-4 competes with Net4 binding to laminin (means.d.; n 3; *P 0.035; ns, not signicant). Error bars, s.d. (n 3 independent cell

cultures). (c) Treatment of VEGF-A induced spheroid sprouting of HDMECs embedded in collagen I by Net4-DC (1 mM). Analysis of sprouting events (ns, not signicant). (d) Tube formation analyses of co-cultured endothelial and perivascular-like cells from untreated and Net4-FL- or Net4-FLE195A,R199A-treated

cultures (30 nM). Representative images of CD31 staining are shown. The tube length (mm per mm2) was quantied through the CD31 staining (means.d.; n 5; ****Po0.00005). (e) Apotome images of Col-IV, Lmg1 and CD31 stained tubes from control (ctrl), Net4-FL and Net4-FLE195A,R199A treatment are

displayed. Error bars, s.d. (n 3, (ac); n 5, (d,e) independent cell cultures). P values, two sided t-test. Scale bars, 100 mm (ad); 50 mm (e).

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Figure 7 | Laminin polymers are essential to maintain capillary networks and tumour progression. (a) Visualization of capillaries via FITC-dextran injection of untreated (ctrl), Net4-FL- and Net4-FLE195A,R199A-treated CAMs after 48 h. The vascularized area from different protein treatments (left) was

determined (see Methods section) at 48 h (means.d.; n 5; ****Po0.0001). Error bars, s.d. (n 5 independent cell cultures). (b) H&E staining of

untreated (ctrl) and Net4-FL-, and Net4-FLE195A,R199A-treated CAMs after 48 h. Red lines and red arrow heads indicate the capillary density (capillaries are

labelled with C). (c) Diagram showing the tumour treatment regimen. (d) Macroscopic images of melanoma treated daily with 1 mM of control protein (mouse serum albumin, left), Net4-FL (middle), Net4-FLE195A,R199A (right). (e) Progression of melanoma treated with Net4-FL and the laminin g1 binding mutant Net4-FLE195A,R199A (means.d.; n 5; ****Po0.0001). Error bars, s.d. (n 5 animals, female C57BL/6). P values, two sided t-test. Scale bars,

100 mm (a); 20 mm (b); and 5 mm (d).

In the vasculature, laminin disassembly destabilizes three-dimensional endothelial tubes in an endothelial/pericyte-like cell co-culture via interfering with the formation of a continuous BM along endothelial cell surfaces. Vessel maturation is affected by establishing a continuous BM along endothelial tube-linings. Cooperation between endothelial (ECs) and PVCs induces the production of pivotal matrix components such as nidogen-1, linking the collagen IV structure to the laminin network, and the a5-chain shaping laminin 511, which is the predominant laminin heterotrimer in mature capillaries28. The nding that treatment of an EC/PVC co-culture and chicken CAMs with Net4 results in decreased vascularized areas further highlights the essential role of vascular laminin networks in angiogenic processes. Furthermore, the Net4 laminin-binding mutant showed no effect on angiogenesis. Remarkably, Net4 disrupts the entire vBM lying between the endothelium and pericytes as well as the BM surrounding pericytes inhibiting stabilization of the capillary network and formation of the common vessel hierarchy ex ovo. Mural cells embedded in the vBM are dened as pericytes29. Upon their loss, as demonstrated by ultrastructure analyses of

Net4-treated CAMs, the phenotype of pericytes is altered to a more mural cell character. These ndings are of potential importance in modulation of cancer progression where blood vessels are not always well organized, but appear destabilized and are often leaky, which hampers drug delivery and increases tumour cell spread4951. The complex between laminin g1 and

Net4 clearly hinders the formation of, or even disrupts, the laminin network. Interestingly, disruption of the vBM leads to detachment of pericytes from the endothelium resulting in the collapse of the entire capillary network (model pictured in Fig. 8c), which may also help to reduce tumour progression. A recent study showed increased permeability in lymphatic vessels upon Net4 overexpression21, which ts with the proposed model. Of interest, this model supports the view that the impact of Net4 depends highly on its ability to titrate laminin and thus an important aspect that remains to be investigated further in animal models is how physiological levels of Net4 actually modulates basal membranes and how in pathological conditions, up- or downregulation of Net4 directly affects the BM and thus the cell behaviour in this Net4 high/low environment. Several studies

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(m) 6050403020

Net4-FLE195A,R199A

Net4-FL

Vessel hierarchy

Vessel diameter (m)

Net4-FLE195A,R199A

Net4-FL

15 30 Length: 100 500

45 60

30 45

3 6

7 10 Length: 30 - 70

28 37

10 20

n.p.

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Pericyte

Endoth.

+ Net4

vBM

Collagen IV

Net4

Laminin

LN LN

LN LN Net4

LN LN

Anchorage

Integrin

SGLs

Figure 8 | Impact of Net4 on disruption of the laminin network and its effect on vascularization. (a) Diagram indicates vessel diameter distribution (left) within Net4-FLE195A,R199A-treated CAMs (blue squares), and the Net4-FL group (red triangle). Model showing the common established capillary hierarchy

formed within the CAM. Numbers indicate sizes of different vessels in the common established hierarchy (middle). The table displays the diameter of different hierarchical vessels from Net4-FLE195A,R199A- (blue) and Net4-FL-treated (red) experiments (right) (n.p., not present). (b) Ultrastructural analyses of Net4-FLE195A,R199A- (blue) and Net4-FL-treated (red) CAMs using transmission electron microscopy. Images show the capillary endothelium surrounded by perivascular cells. The endothelium E is shown and PVCs are indicated as P. Electron microscopy images highlight the vascular basement membrane (vBM, red arrow heads (middle) and red asterisks (bottom)) between the endothelium and PVCs, which are surrounded by a vBM layer. (c) Model of Net4 effect on capillary cell types (endothelium (Endoth.) and pericyte (Pericyte)) and the vBM. Scale bars (b), 2 mm (top); 1 mm (middle); 0.5 mm (bottom).

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have shown that Net4 expression is modulated in cancer and as an example is inversely associated with cancer progression52 suggesting that Net4 could represent an important modulator of tumoral matrix and thus a putative therapeutic target. Finally, our study not only highlight the key role of laminin networks within the vBM but also shows that Net4 disrupts pre-existing laminin networks and BMs in a non-enzymatic manner.

Methods

Recombinant protein expression and purication. Net4-FL, g1LN-LEa1-4 and serum albumin from Mus musculus, (Net4: NP_067295, aa: 20-628, g1LN-LEa1-4: NP_034813, aa: 34-492, serum albumin: NP_033784, aa: 1-608) and a1LN-LEa1-4, b1LN-LEa1-4 from Homo sapiens (a1LN-LEa1-4: NP_005550, aa: 18-453, b1LNLEa1-4: NP_002282, aa: 31-509) were cloned into a modied pCEP vector with an N-terminal double Strep II-tag. Net4-DC from Mus musculus (NP_067295, aa: 20-462) was cloned into a modied pCEP vector with an N-terminal octa-histidine (His8) tag or with an N-terminal double Strep II-tag. HEK293 cells were stably transfected followed by screening for subclones with a high level of protein expression. Net4-DC, Net4-FL, g1LN-LEa1-4, a1LN-LEa1-4, and b1LN-LEa1-4 were puried by streptactin-sepharose (IBA) and Net4-DC by metal-afnity chromatography using nickel TALON beads (Clontech) followed by the removal of the double Strep II-tag and His8-tag by thrombin digestion. The puried proteins were then dialyzed against 1 x PBS. Afnity puried proteins were further processed by size-exclusion chromatography (SEC) in TBS buffer (50 mM Tris-HCl, pH 7.5; 200 mM NaCl) on a Superdex 200 column using theKTA FPLC system (GE Healthcare Life Sciences USA) equipped with UV absorbance detector and UNICORN v5.11 software as previously described. The complex of Net4-FL-g1LN

LEa1-4 and Net4-DC-g1LN-LEa1-4 was puried using SEC by mixing the peak fractions for individual species. Protein concentrations were measured using extinction coefcient and molecular weight values derived from the ProtParam utility53 available on the ExPaSy server54.

Design of chimeric constructs and site-directed mutagenesis. To design Net4 containing LNg1 binding domains we generated chimeric molecules by overlap PCR using the Q5 (New England Biolabs) polymerase. The domains from Net4 (Net4: NP_067295) were replaced by the corresponding laminin g1 sequences (laminin g1: NP_034813). The following constructs were amplied, cloned (with an N-terminal double Strep 2-tag), veried by sequencing, expressed and puried: Net4LN-LE1-2g1LEa3-4 (Net4 aa: 20-394 fused to laminin g1 aa: 396-492),

Net4LN-LE1g1LEa2-4 (Net4 aa: 20-331 fused to laminin g1 aa: 340-492), g1LNLEa1Net4LE2-31/2 (Laminin g1 aa: 44-339 fused to Net4 aa: 332-462) and Net4-DCN-term agrin (NP_067295, aa: 20-462, E195A, R199A, fused to NP_067617, aa: 33164). Furthermore, the following mutated Net4 molecules were generated: Net4-FL: (NP_067295, aa: 20-628, E195A,R199A), Net4-DCE195A,R199A,DKAPGA: (NP_067295, aa: 20-462, E195A, R199A and D281KAPGA285), Net4-DCE195A,R199A: (NP_067295, aa: 20-462, E195A, R199A and D281KAPGA285), and Net4-DCE195A,R199AN-term agrin (NP_067295, aa: 20-462, E195A, R199A, fused to NP_067617, aa: 33164). All produced proteins are shown in Supplementary Fig. 12.

Structure determination of Net4. Net4-DC was applied to a Superdex 200 SEC column (GE Healthcare) equilibrated with 50 mMTris/Tris-HCl, pH 7.5, 0.2 M NaCl at room temperature and the collected peak fractions were immediately concentrated to 1.2 mg ml 1 and used for crystallization. Net4-DC crystals were grown by hanging-drop vapour diffusion at 293 K by mixing 2 ml of protein solution, 1.6 ml of reservoir solution (20% PEG3350, 0.2 M calcium acetate, and0.1 M sodium cacodylate pH 6.5), and 0.4 ml of 1 M NDSB-256 from the Hampton HR2-428 additive screen (Hampton Research). Crystals appeared after 1 week and were soaked in mother liquor containing 10% ethylene glycol for 510 min before ash freezing in liquid nitrogen. Diffraction data was collected on a Rigaku MM-007HF (l 1.54178 ) at 100 K. The data set was indexed, integrated and scaled

with the HKL2000 suite. The phases were determined by molecular replacement using a polyserine laminin-b homology model (PDB4AQS55) from Swissmodel in

Phaser56. The model was built and rened using Coot57, the Phenix Rene and Autobuild programs58 and Refmac59,60.

Octet-binding studies. Binding of Net4 or Net1 to DCC, neogenin, and UNC5H2 was analysed by biolayer interferometry using the Octet RED96 system (Pall ForteBio). Recombinant human DCC/Fc, mouse neogenin/Fc and rat UNC5H2/Fc chimera were obtained from Bio-Techne. Proteins were stored frozen and diluted into binding buffer (PBS, 0.1% BSA, 0.02% Tween 20). Binding assays were performed in 96-well microtitre plates at 30 C with orbital sensor agitation at 1,000 r.p.m. Fc chimera proteins (2 mg ml 1) were loaded onto anti-human Fc (AHC) biosensors for 10 min. Biosensors were washed with binding buffer for 60 s and placed in wells containing the various netrins (50 nM in binding buffer). Association was observed for 10 min, then biosensors are incubated in binding buffer for additional 10 min to observe dissociation of the complex. Uncoated

biosensors were run in parallel for non-specic binding. Binding curves were analysed by Octet User software (ForteBio). Three independent technical replicates were performed.

Microscale thermophoresis binding assay. Binding of the labelled Laminin g1 short arm fragment (g1LN-LEa1-4) to Net4 variants and its respective mutants were measured using microscale thermophoresis. A range of concentrations of the required ligands (from 0.08 to 1,000 nM for Net4 and from 0.45 to 4,000 nM for Net4 mutants) were incubated with 2 nM of g1LN-LEa1-4 in the assay buffer(50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2 supplemented with 3% BSA) for 10 min. Samples were loaded into NanoTemper glass capillaries and microscale thermophoresis was carried out using 10% light-emitting diode power and 80% MST power on a NanoTemper monolith NT.115 (ref. 61). KD values were calculated from independent technical triplicate experiments using the mass action equation via the NanoTemper software.

Laminin network disruption assay. The laminin polymerization-blocking assay was performed as described in the Supplementary Fig. 9b and the laminin polymer disruption assay setup is described in the Supplementary Fig. 9c. Mouse laminin 111 (Sigma-Aldrich, Germany) was diluted to 0.35 mM in 50 mM Tis-HCl, pH 7.4;

150 mM NaCl; 0.1% Triton-X100; 1 mM CaCl2 and incubated at 37 C for 3 h to polymerize. Afterwards, polymerized laminin was centrifuged (10,000g; 15 min) to separate formed laminin polymer from non-polymeric laminin. Different proteins (Net4-DC, Net4-DCE195A,R199A) were added at a concentration of 2.1 mM respectively to the laminin polymer at 37 C for additional 3 h. Samples were centrifuged and all three fractions (supernatant (S), supernatant (S ), pellet (P );

Supplementary Fig. 7b) were analysed by SDSPAGE (SDSpolyacrylamide gel electrophoresis) followed by fast Coomassie Blue staining (Thermo Scientic, Germany). Band intensities were determined using the ImageJ software and ratios between the polymer fraction (P ) and supernatant (S ) are displayed.

Laminin assembly on Schwann cell surface. Rat Schwann cells were cultured in Dulbeccos Eagle medium, 10% fetal calf serum (Atlanta Biological), neuregulin(0.5 mg ml 1, Sigma), forskalin (0.2 mg ml 1, Sigma) and penicillin-streptomycin. Cells at passage 22-29 were seeded onto 24-well dishes (Sigma) and incubated with collagen IV, nidogen-1 and different laminin constructs. Additionally, cells were treated with 28-fold excess compared with laminin of Net4-FL, Net4-FLE195A,R199A at 37oC for 1 h. Schwann cells were rinsed with PBS and xed in 3% paraformaldehyde for 20 min followed by blocking with 5% goat serum in PBS overnight at 4 C. Cells were incubated with anti-laminin g1 (1:200, Millipore, MAB1920)

and anti-collagen IV (10 mg ml 1, Millipore, AB756P) at room temperature for 1 h. Alexa Fluor 647 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit secondary antibodies (Molecular Probes) were used at 1:100 and counterstained with 40,6-diamindino-2-phenylindol. Digital images of protein immunouorescene levels were recorded with IPLAB 3.5 software (Scanalytics) and quantied with IMAGE J. Validation information for commercial antibodies is available on the manufacturers websites. All experiments were performed in groups of ve independent cultures and repeated at least twice.

Neurite outgrowth assay. Olfactory bulb (OB) explants were isolated from rat embryos E15 (n 19 Embryos at E15 were collect from pregnant Sprague Dawley

female rat). OB explants were embedded in Collagen I isolated from rat tail and cultured with 28 nM of Net4-FL or Net4-FLE195A,R199A for 24 h. Explants were then xed in 4% PFA at room temperature for 1 h and permeabilized with PBST (PBS, 0.1% Triton-X100) for 10 min. Next, explants were blocked with PBST, 1% normal donkey serum (Jackson Immunoresearch) at room temperature for 2 h and incubated with mouse polyclonal Tuj1 antibody, 1/500 (Babco, MMS435P) diluted in PBST at 4 C overnight. After 3 times washing with PBST, explants were incubated with Alexa488 donkey anti mouse (1/500; Life Technologies, A21202) at room temperature for 1 h, followed by 3 washes in PBST and mount with mowiol. Explants were imaged with Axiovert200M, Zeiss. Neurite and explant area were analysed with FIJI in order to determine neurite density (neurite area/explant area). Validation information for commercial antibodies is available on the manufacturers websites. All experiments were performed in groups of ve independent cultures and repeated at least twice.

Cell culture. HEK293 EBNA cells as well as the mouse melanoma B16-F1cell line were obtained from ATCC. HDMEC and HDLEC were obtained from Promocell; HUVEC from Lonza. The endothelial cells were cultured in Vasculife VEGF-Mv Medium Complete Kit (Cell Systems) according to the manufactures instructions. The mouse melanoma cell line (HCMel12) was kindly provided byT. Tting (University of Magdeburg)62. The mouse melanoma cell lines HCMel12 and B16-F1 as well as HEK293 EBNA cells were cultured in Dulbeccs Modied Eagle Medium (Gibco) supplemented with 10% fetal bovine serum. PVCswere isolated from meninigi of the Anxa5-LacZ reporter mouse strain63.

PVCs were cultured in DMEM supplemented with 10% FCS and used at passages 2840.

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Tube-like formation assay. Cells were seeded at a density of 25,000 cells per well onto chamber slides (Labtek) or ibidi slides (ibidi) previously coated at 37 C with Matrigel BM matrix (Corning, New York, USA) for 30 min to allow polymerization. For the tube formation assay, the indicated proteins were added directly after seeding the cells. Cells were incubated for 14 h. For the tube regression assay proteins were added 7 h after seeding the endothelial cells, which had established a tubular network. Recombinant proteins were added at the concentrations indicated and in terms of endothelial cells treatment PDGF as well as VEGF (Biomol) was added at nal concentrations of 10 ng ml 1. Tube formation was documented by bright-eld microscopy using a Nikon Eclipse TE2000-U microscope. In order to investigate the time course of tube formation, we performed time-lapse microscopy using an Olympus IX81. All experiments were performed in groups of ve independent cultures and repeated at least three times.

Three-dimensional culture in collagen I matrix. Endothelial and pericyte-like cells (HUVECs/PVCs) were co-cultured in 2% collagen I gel. Cultures were incubated in ECGM2 medium supplemented with PDGF-BB and VEGF (10 ng ml 1 each) for 6 days. Recombinant Net4-FL and Net4-FLE195A,R199A were added to the three-dimensional gel solution at indicated concentration. Medium (supplemented with PDGF-BB, VEGF, and Net4-FL or Net4-FLE195A,R199A) was replaced every 48 h. Samples were xed with Dents xative and immunostained for collagen IV (1:500; AB769, Chemicon), laminin g1 (1:500; kind gift from U. Mayer, University of East Anglia), and human CD31 (1:500; 555444, BD Biosciences). DNA was stained by Hoechst 33258. Overview pictures were taken from each sample by using a Zeiss Axioplan microscope and average tube length was calculated by the Volocity software. Optical section images were taken by a Zeiss Apotome microscope and processed by Axiovision program. All experiments were performed in groups of ve independent cultures and repeated at least three times. Validation information for the laminin g1 antibody is presented in the work of Mayer et al.64

Chick chorioallantoic membrane angiogenesis assay. The ex ovo culture of the chicken embryos was adapted from Auerbach et al.46 Briey, fertilized Ross chicken eggs (Wthrich Brterei AG, Switzerland) were incubated at 37 C for 3 days in humidied atmosphere. On day 3, the eggs were carefully opened and their content was transferred into 100 20 mm Petri dishes (Corning, Switzerland). The

chicken embryos were incubated in the same conditions for 6 more days. On day 9, the polyethylene glycol (PEG) hydrogels containing no protein and different proteins (Net4-FL, Net4-FLE195A,R199A) were placed on the surface of the CAM. 15 ml PEG hydrogels were formed by Factor XIIIa-catalysed crosslinking of two eight-arm PEG components (8-PEGGln and 8-PEGMMPsensitiveLys) in 50 mM

Tris pH 7.6, 50 mM CaCl2 buffer to a nal PEG concentration of 2% (w/v). Each hydrogel contained 4.25 mg of recombinant Net4-FL, or Net4-FLE195A,R199A, which were added to the hydrogel mixture before polymerization. After 48 h of treatment, 100 ml of 2.5% FITC-dextran (MW 20,000, Sigma-Aldrich, Switzerland) in 0.9%

NaCl solution were injected intravenously in the CAM. The vasculature was visualized by uorescence microscopy and images around the treatment site were collected. All experiments were performed in groups of ve independent cultures and repeated at least twice.

The images collected by uorescence microscopy were analysed using the ImageJ software (ImageJ 1.48, http://imagej.nih.gov/

Web End =http://imagej.nih.gov/ ) using a script based on the quantication method developed by Blacher et al.65 Background of each image was evened out and the contrast was enhanced. Then the threshold was adjusted manually do discriminate the vascularized area from the non-vascularized area, and the image was transformed into a binary image. A series of lters were applied then to rene the capillary structures. Region of interests (ROIs) were set manually to exclude second-order and higher-ranking blood vessels and regions with underlying blood vessels. The vascularized areas within the ROIs, as well as the total ROI area, were measured. The obtained values were used to determine the distribution vascularized areas in the capillary network. All experiments were performed in groups of ve independent cultures and repeated at least three times.

Immunohistochemistry of the CAM. After live imaging, the CAMs were xed with 4% para-formaldehyde solution by applying the solution both on top and beneath the CAM membranes and incubated at room temperature for 1 h. The area around the treatment site was carefully cut out and placed between cellulose sheets before being processed for parafn embedding. Tissue sections of 4 mm were cut with a microtome and mounted on glass slides for haematoxylin and eosin staining.

Transmission electron microscopy analyses of CAM capillaries. After live imaging, CAMs were xed with 2.5% glutaraldehyde (buffered with 0.1 M sodium-cacodylate, pH 7.4, 540 mOsm.) by applying the solution both on top and beneath the CAM membranes. They were post-xed in 1% OsO4 (0.1 M sodium-cacodylate, pH 7.4, 340 mOsm), dehydrated in ethanol and embedded in Epon 812 (Fluka, Buchs, Switzerland). Semi-thin sections were prepared, stained with toluidine blue, and analysed with a light microscope66,67. Representative areas were selected and further imaged by transmission electron microscopy. Tissue samples from groups of three of three independent biological replicates were analysed.

Mice model of melanoma. The melanoma HCmel12 cell line, which spontaneously develops lung metastases after intracutaneous injection in immuno-competent C57BL/6 mice, was established from a primary Hgf-Cdk4R24C melanoma62. For tumour transplantation 2 105 cells in 100 ml 1 PBS were

injected intracutaneously into the anks of C57BL/6 mice (female, n 5, 8 weeks of

age) and tumour development was monitored by visual inspection and palpation. When tumours exceed 2 mm groups of ve mice were peritumorally injected daily with 1 mM of mouse albumin (ctrl), Net4-DC, Net4-FL and Net4-FLE195A,R199A for 10 consecutive days. Tumour sizes were measured every second day and recorded as mean diameter in mm. Mice with tumours exceeding 20 mm diameter were killed. All experiments were performed in groups of ve mice and repeated independently at least twice.

Statistics. Statistical analysis was performed using the GraphPad Prism software (GraphPad Prism 5.00). The two sided t-test was used for statistical analysis, statistical analyses of the Schwann cell assay was performed using the one-way analysis of variance followed by a pairwise comparison using the HolmSidak method. Results are displayed as means.d. except the one of the neurite outgrowth assay, in which the results are displayed ass.e.m.

Study approval. All animal experiments were performed in accordance with the German laws for animal protection and were approved by the State Ofce North Rhine-Westphalia (LANUV), Germany or by the CECCAP, France.

Data availability. The data that support the ndings of this study are available from the corresponding authors upon request. Coordinates and structure factors for Net-4DC have been deposited in Protein Data Bank under accession code 4WNX.

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Acknowledgements

We thank Jan Zamek, Birgit Blck, Thomas Dietz and Werner Graber for excellent

technical support. Further, we thank Dr Michael Lammers for the opportunity to use the

Monolith NT.115 Series (NanoTemper). This work was supported by grants from the

DFG (A2, CRC829) to M.K. and from ANR, INCA, ERC, Ligue contre le Cancer to P.M.

J.S. holds a Canada Research Chair in Structural Biology and this investigation was

supported in part from the Multiple Sclerosis Society of Canada and by the Canadian

Institute of Health Research (RPA-109759). T.R.P. was funded by Canadian Institutes of

Health Research Postdoctoral Fellowship and Marie Skodowska-Curie Fellowships.

M.McD. was funded by grants from the CIHR Operating Grant. M.E. was supported by

funding from the Swiss National Science Foundation (Nos. CR3213-125426/1 and

310000-116240). T.T. was supported by the DFG (A22, CRC704) and Deutsche Kreb

shilfe (P9 in the Melanoma Research Network). P.Z. was funded by the DFG (B4,

CRC829) and Deutsche Krebshilfe (P4 in the Melanoma Research Network). P.D.Y. was

supported by a grant from National Institutes of Health (NIH), grant R01-DK36425. C.P.

and H.C.-S. acknowledge the nancial support from the CANTER For

schungsschwerpunkt of the Bavarian State Ministry for Science and Education from the

DFG (grant CL 409/2-1).

Author contributions

R.R. discovered the underlying mechanism of Net4 as well as designed and coordinated

the experiments, produced recombinant proteins, performed biochemical experiments,

analysed, and visualized the data. T.R.P. and M.M. performed further protein purica

tion, for Net4-DC, Net4-FL and g1LN-LEa1-4 as well as puried their complexes. T.R.P.

performed biophysical studies. T.R.P. initiated crystallization studies and identied

conditions. M.McD. performed the data collection and solved the X-ray structure. D.N.

performed and analysed SPR measurements. J.-G.D. performed Octet binding analysis.

M.M. and M.McD. performed AUC experiments and T.R.P. analysed the data. C.P.,

H.C.-S. and R.R. performed the AFM measurement of the Matrigel matrix. C.P. analysed

the data. Z.Z. and E.P. performed the EC/PVC co-culture assay. S.M. and M.E. per

formed CAM studies. N.R. and B.G. performed the OB explant assay. R.R., T.B., T.T. and

P.Z. performed tumour treatment assays. V.D. performed TEM analyses. K.K.M. and

P.D.Y. performed BM assembly assay. W.B. isolated endothelial cells from EBs and

supported EM analysis. P.D.Y. and R.R. designed the Net4 mode of action model. R.R.

designed experiments. R.R., P.M, M.K. and J.S. conceived and supervised all aspects of

the project and interpreted the data. R.R., T.R.P., M.McD., M.M., P.M., J.S. and M.K.

wrote the manuscript. All authors reviewed the manuscript.

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Abstract

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Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.

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Title

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes

Author

Reuten, Raphael; Patel, Trushar R; Mcdougall, Matthew; Rama, Nicolas; Nikodemus, Denise; Gibert, Benjamin; Delcros, Jean-guy; Prein, Carina; Meier, Markus; Metzger, Stéphanie; Zhou, Zhigang; Kaltenberg, Jennifer; Mckee, Karen K; Bald, Tobias; Tüting, Thomas; Zigrino, Paola; Djonov, Valentin; Bloch, Wilhelm; Clausen-schaumann, Hauke; Poschl, Ernst; Yurchenco, Peter D; Ehrbar, Martin; Mehlen, Patrick; Stetefeld, Jörg; Koch, Manuel

Pages

13515

Publication year

2016

Publication date

Nov 2016

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms13515

ProQuest document ID

1844732844

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes

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