Coordination of VEGF receptor trafficking and signaling by coreceptors

E XP E RI ME N T AL C E L L RE S E AR CH

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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Review Article

Coordination of VEGF receptor trafficking and signaling by coreceptors Masanori Nakayamaa, Philipp Bergerb,n a

¨ nster, Germany Max-Planck-Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, D-48149 Mu Paul Scherrer Institute, Biomolecular Research, Molecular Cell Biology OFLC 101, CH-5232 Villigen PSI, Switzerland

b

article information

abstract

Article Chronology:

During development, regeneration and in certain pathological settings, the vasculature is

Received 20 November 2012

expanded and remodeled substantially. Proper morphogenesis and function of blood vessels

Received in revised form

are essential in multicellular organisms. Upon stimulation with growth factors including

25 February 2013

vascular endothelial growth factors (VEGFs), the activation, internalization and sorting of

Accepted 2 March 2013

receptor tyrosine kinases (RTKs) orchestrate developmental processes and the homeostatic maintenance of all organs including the vasculature. Previously, RTK signaling was thought to

Keywords:

occur exclusively at the plasma membrane, a process that was subsequently terminated by

VEGFR2

endocytosis and receptor degradation. However, this model turned out to be an oversimplifica-

Neuropilin

tion and there is now a substantial amount of reports indicating that receptor internalization

VE-cadherin

and trafficking to intracellular compartments depends on coreceptors leading to the activation

Ephrin

of specific signaling pathways. Here we review the latest findings concerning endocytosis and intracellular trafficking of VEGFRs. The body of evidence is compelling that VEGF receptor trafficking is coordinated with other proteins such as Neuropilin-1, ephrin-B2, VE-cadherin and protein phosphatases. & 2013 Published by Elsevier Inc.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VEGFR2 activation and initiation of intracellular trafficking . . . . . . . . Influence of NRP1 on VEGFR2 trafficking. . . . . . . . . . . . . . . . . . . . . . . Signaling crosstalks between notch and VEGF at angiogenic sprouts . Protein turnover during vessel formation in angiogenesis . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Activation, internalization and sorting of membrane receptors plays a crucial role in the development and maintenance of all

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organisms [1]. Altered trafficking of mutated membrane proteins or defects in the trafficking machinery lead to aberrant protein processing and signaling, which is indeed observed in many human diseases [2]. Furthermore, intracellular trafficking defects

n

Corresponding author. Fax: þ41 56 3105288. E-mail address: [email protected] (P. Berger).

0014-4827/$ - see front matter & 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.yexcr.2013.03.008

Please cite this article as: M. Nakayama, P. Berger, Coordination of VEGF receptor trafficking and signaling by coreceptors, Exp Cell Res (2013), http://dx.doi.org/10.1016/j.yexcr.2013.03.008

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of transmenbrane proteins can cause several problems. A prominent example is the processing of amyloid precursor protein (APP) to its non-amyloidogenic form at the plasma membrane by a-secretase and to its amyloidogenic form in endosomes where APP encounters b- and g-secretases [3]. Vascular endothelial growth factor receptors (VEGFR1/2/3) are receptor tyrosine kinases that play crucial roles in blood and lymphatic vessel development and maintenance and under pathological conditions. They are activated by five different VEGFs (VEGF-A, -B, -C, D, and placenta growth factor) that all exist in multiple isoforms (reviewed by [4]). The VEGF-A/ VEGFR2 signaling complex is the major regulator and required for almost every aspect of blood vessel formation and function. Nevertheless, the resulting plethora of VEGF/ VEGFR combinations is still not sufficient to fulfill all biological functions. Therefore, VEGFRs dynamically associate with coreceptors such as Neuropilin or ephrin-B2 that guide receptors through the cell where they come in contact with other factors such as phosphatases or kinases. Here we summarize how VEGFR containing protein clusters regulate blood vessel development and maintenance.

VEGFR2 activation and initiation of intracellular trafficking VEGFR2 is predominantly expressed in endothelial cells. Targeting of Flk1, the gene encoding VEGFR2, causes impaired development of endothelium and embryonic lethality at E8.5 [5]. Deletion of a single allele of Vegfa, encoding VEGF-A, leads to a similar phenotype, suggesting an essential role of the VEGFR2/ VEGF-A system in vessel development [6,7]. In vitro, approximately 50% of the VEGFR2 of an endothelial cell is exposed at the plasma membrane whereas the other half localizes to endosomal vesicles, indicative of constitutive turnover under resting conditions [8]. The delivery of VEGFR2 from the Golgi to the plasma membrane occurs in a syntaxin-6 dependent manner [9]. On the plasma membrane, a fraction of the VEGFR2 clusters with caveolin. These clusters dissociate rapidly after stimulation with VEGF-A and internalization probably occurs in a caveolinindependent fashion [10]. VEGF-A induces homodimerization, or heterodimerization of VEGFR2 with VEGFR3 or VEGFR1 and activation of these tyrosine kinases through transphosphorylation, leading to the initiation of intracellular signaling cascades [11,12]. The extracellular part of VEGFR2, consisting of seven Ig-like domains, is flexible in its monomeric form. After binding of ligands, they form a rigid and intertwined dimer [13,14]. The majority of VEGFR2 and presumably VEGFR3 are internalized by clathrin-mediated endocytosis. Internalization does not terminate signaling; rather downstream signals are further generated from the plasma membrane as well as from endocytic vesicles. The signaling potential of tyrosine phosphorylation of VEGFRs has recently been reviewed by [15] and is therefore not discussed in detail here. VEGFR2 can be inactivated by dephosphorylation (see below) or degradation [16,17]. Ubiquitination and E3 ubiquitin ligases are known to play important roles in protein degradation. Several ubiquitin ligases are known to be involved in VEGFR2 degradation and attenuation of signaling. Activation of VEGFR2 induces phosphorylation of the ubiquitin ligase Cbl leading to polyubiquitination of VEGFR2 and its degradation [18]. In addition, Cbl regulates the activity of PLCg1, a downstream target of VEGFR2, by a direct interaction [19]. Phosphorylation of a cytoplasmic PEST motif of VEGFR2 leads to recruitment of the E3

] (]]]]) ]]]–]]]

ubiquitin ligase b-Trcp1 which polyubiquitinates VEGFR2 leading to its degradation [20]. Another regulator of VEGFR2 degradation is Nedd4, a HECT domain-containing ubiquitin ligase. It acts indirectly on VEGFR2 levels by associating with the VEGFR2 adapter protein Grb10 [17]. However, VEGFR2 appears to undergo both lysosomal and proteasomal-mediated degradation in endosomes and lysosomes [21]. VEGFR ubiquitination is also involved in VEGFR endocytosis. Epsin1 and 2, two ubiquitin-binding endocytic clathrin adapter proteins, are involved in tumor angiogenesis. Loss of Epsin1/2 compromises VEGFR2 endocytosis, resulting in excessive VEGF signaling [22]. Taken together, many processes were discussed for the inactivation of VEGFR2 suggesting that the cellular context plays an important role.

Influence of NRP1 on VEGFR2 trafficking Neuropilins were initially identified as receptors for class-3 semaphorins that are involved in axon guidance [23,24]. Subsequently, it was found that neuropilins also interact with VEGF-A [25]. Neuropilin-1 knock out mice are able to form blood vessels but the vascular network is severely disorganized and the mice die at E12.5 [26]. VEGF-A165a can simultaneously bind to VEGFR2 and NRP1 even when they are expressed separately on adjacent cells. Binding of VEGFs to NRP1 is mediated by the C-terminal motif of VEGFs that consists of prolines and basic amino acids [27]. Interestingly, alternative splicing of the terminal exon 8 of VEGF-A leads to two different isoforms; the NRP1-binding a-series (e.g. VEGF-A165a) and the non-NRP1-binding b-series (e.g. VEGF-A165b) [28]. VEGF-A165b is a weak agonist of VEGFR2 compared to VEGF-A165a and it fails to induce vessel formation in a sprouting assay in vitro [29]. The administration of VEGF-A165b to mice reduces tumor growth with no side effects in other tissues [30]. An antiangiogenic potential was also observed in a neovascularization experiment in the eyes of mice [31]. In the meantime, the relevant splicing factors that influence the ratio between the a- and b-forms were identified [32,33]. Receptors are either internalized constitutively or upon stimulation with a ligand. VEGFR2 colocalizes with the endosomal markers Rab5, Rab4, and hrs, but not with Rab11 [16,34,35]. NRP1 recycles in contrast to VEGFR2 through RAB11 vesicles back to the plasma membrane [36]. The transition from RAB4 to RAB11 vesicles depends on the C-terminal PDZ-binding motif that binds to Synectin/ GIPC1 [37]. Activation of VEGFR2 with VEGF-A165a leads to recruitment of NRP1 as a coreceptor and recycling through the RAB5-RAB4-RAB11 axis and results in reduced degradation (Fig. 1). This recycling route is associated with enhanced p38 signaling and vessel formation in in vitro assays [36,38]. Synectin/ GIPC1 binds to Myosin VI, a well known minus-end directed actin motor involved in endocytosis. Deletion of Gipc1, encoding Synectin/ GIPC1, in mice or reduction of the expression level in zebrafish led to vascular defects similar to the neuropilin-1 knock out, suggesting that VEGF-A165a/ VEGFR2/ NRP1/ GIPC1/ Myosin VI functionally interact in vivo [39,40]. Interestingly, mutant mice that express neuropilin-1 lacking the cytoplasmic domain are viable. The vasculature appears nearly normal, only an enhanced number of arterial/ vein crossings in the retina were observed. The interaction of the cytoplasmic domain of NRP1 with Synectin/ GIPC1 might therefore be important for arterial maturation or endothelial functions such as vascular permeability rather than for angiogenesis [41] or other receptor systems can compensate for the lack of NRP1.

Please cite this article as: M. Nakayama, P. Berger, Coordination of VEGF receptor trafficking and signaling by coreceptors, Exp Cell Res (2013), http://dx.doi.org/10.1016/j.yexcr.2013.03.008

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a

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Recycling

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Rab5

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Rab4 a

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Degradation

VEGFR-2

P Phosphorylation

GIPC

NRP-1

a VEGF-A165a

Myosin-VI

Actin

unidentified phosphatase

PTP1b

Fig. 1 – NRP1 as a coreceptor of VEGFR2. VEGFs induce dimerization and autophosphorylation of VEGFR2. VEGFA165a is in contrast to VEGF-A165b able to recruit NRP1 as a coreceptor. NRP1 links VEGFR2 via GIPC to Myosin-VI, a minus-end directed actin motor. This brings VEGFR2 to the Rab11 recycling compartment. The molecular mechanism of the transport to the plasma membrane and the integration site into the plasma membrane is not known. During trafficking, VEGFR2 is dephosphorylated by at least two phosphatases. PTP1B dephosphorylates VEGFR2 near the plasma membrane and a so far unidentified phosphatase between RAB4 and Rab11.

Whereas VEGFR2 is not dephosphorylated for degradation, NRP1dependent recycling of VEGFR2 through RAB11 vesicles is associated with its dephosphorylation at Y1175 and Y1212 (other phosphotyrosines were not tested) by an unidentified phosphatase. This trafficking route also leads to enhanced p38 signaling which is in line with the observation that activation with the NRP1-binding VEGF-A165a leads to higher P-p38 levels than activation with VEGFA165b [36,38]. This suggests that VEGFR2 is reintegrated into the plasma membrane at specific sites for a second round of activation which then leads to prolonged signal output. For example, it was shown that association of VEGFR2 with b1-integrin at focal adhesion leads to prolonged p38 activation [42] and that NRP1 is necessary for the phosphorylation of focal adhesion kinase [43]. Several non-receptor tyrosine phosphatases with different specificities for tyrosines act on endosomal VEGFR2. Unfortunately, their specificities, their effects on downstream signaling, and their localization were not systematically analyzed so far. Lanahan et al. showed that delayed trafficking due to GIPC1-myosin VI blockage leads to prolonged exposure to PTP1b that then dephosphorylates VEGFR2 at Y1175 at or near the plasma membrane [44]. PTP1b overexpression selectively reduces Erk but not p38 activation indicating that it is not involved in dephosphorylation of VEGFR2 between Rab4 and Rab11 vesicles [45].

Signaling crosstalks between notch and VEGF at angiogenic sprouts Ephrin-B2, a transmembrane ligand for Eph receptors, possesses intrinsic signaling capabilities that are required for embryogenesis, vasculogenesis, angiogenesis and lymphangiogenesis. Gene inactivation of Efnb2, the gene encoding ephrin-B2, in mice

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compromised vasculogenesis and angiogenesis [46]. Two recent studies highlighted the functional connection of ephrhin-B2 and VEGFR signal transduction. In the absence of ephrin-B2, the internalization of VEGFR was defective in vitro and in vivo, which compromised downstream signaling. Furthermore, ephrin-B2 and its receptor EphB4 induce VEGFR2 and VEGFR3 internalization without downstream signal activation [47,48]. Although molecular mechanisms underlying EphB4-ephrin-B2 induced VEGF receptor endocytosis are largely unknown yet, recent studies have revealed the role of ephrin-B2 in VEGFinduced VEGF receptor internalization. Ephrin-B2 forms a functional complex with the polarity protein PAR-3, the clathrinassociated sorting protein Dab2 and VEGFR2/3. VEGFR2/3 internalization and the activation of downstream signal transduction cascades like small GTPase Rac1 and MAP-kinase are compromised in the absence of Dab2 or PAR-3 whereas PI3kinase pathway is modestly affected [49]. Previous reports show that Dab2 forms protein complex with Synectin/ GIPC [50,51]. VEGFstimulation promotes the interaction of VEGFR2 with NRP1 and Synectin/ GIPC, which is important for VEGFR2 trafficking in vitro as mentioned above. NRP1 and ephrin-B2 therefore might have functional redundancy in vivo. Another key regulator of VEGF receptor endocytosis is VEcadherin-mediated adherens junctions. VEGF-A stimulation induces clathrin mediated VEGFR2 internalization, which is accelerated in VE-cadherin deficient cells [52]. Although most of cell surface exposed VEGFR2 does not localize at cell-to-cell junctions (Nakayama et al. unpublished data), a direct interaction of VEGFR2 with VE-cadherin might be a possible explanation of this mechanism. Consistent with this idea, several works show the immunocomplex of VEGFR2 and VE-cadherin which can retain VEGFR2 at plasma membrane [53–56]. VE-cadherin binding to b-catenin but not to p120-catenin [54], suggests that VEGFR endocytosis is tightly coordinated with junctional remodeling (see below). The sprouting behavior of endothelium is essential for angiogenesis and it is regulated by functional crosstalk between Notch and VEGF/VEGFR signal transduction [57,58]. Notch signaling is a versatile signaling system that controls functions as diverse as cell proliferation, differentiation and tissue patterning in a wide range of organs and animal species [59]. In the developing vasculature, Notch is an important negative regulator of endothelial sprouting and proliferation [58]. In hypoxic tissues, VEGF-A production is upregulated, and tip cells at the leading edge of growing vasculature, sprout by sending out filopodia towards a VEGF gradient. Activation of VEGFR2 leads to upregulation of the Notch ligand Delta-like 4 (Dll4) at tip cells [58]. This, in turn, leads to the activation of Notch receptors in adjacent stalk cells, which form the base of the sprout, and suppresses tip cell behavior in these ECs. Activation of Notch signaling by Dll4 is thought to occur predominantly in stalk cells and downregulates VEGFR gene transcription. Accordingly, Notch is presumably less active in tip cells [58]. However, VEGFR protein expression, in particular, VEGFR2 expression is not higher in sprouts than in more mature vessels [60]. This unexpected finding can be explained by spatially regulated endocytosis of VEGFR. PAR-3 forms a polarity protein complex, called PAR complex, together with PAR-6 and atypical protein kinase C (aPKC), which is a signaling hub of Rho family small GTPases [61]. aPKC phosphorylates Dab2 and negatively regulates VEGFR endocytosis [49].

Please cite this article as: M. Nakayama, P. Berger, Coordination of VEGF receptor trafficking and signaling by coreceptors, Exp Cell Res (2013), http://dx.doi.org/10.1016/j.yexcr.2013.03.008

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The activity of aPKC in the angiogenic front is low, enabling rapid endocytosis. In contrast, higher levels of aPKC activity in the central vascular plexus slow down VEGFR endocytosis and thereby limit downstream signal transduction and degradation. These findings indicate that motile behavior of ECs at the anigogenic front is not exclusively regulated by VEGF-A gradients, but also by a countergradient of VEGF receptor internalization. Thus, spatially regulated endocytosis critically determines endothelial behavior in the growing vasculature [49].

Protein turnover during vessel formation in angiogenesis Adherens and tight junctions play pivotal roles in the developing and in the adult vascular system. In endothelial cells, they are very often intermingled and not clearly separated as in epithelial cells [62]. Adherens junctions in endothelial cells are mainly formed by VECadherin whereas claudin 1, 5, 12 and occludin are the main components of tight junctions. Mice lacking the VE-Cadherin gene or expressing VE-Cadherin with C-terminal truncations die at E9.5. The embryos develop normally until E8 indicating that the assembly of the primitive capillary plexus is not affected, but the remodeling into a network of arteries and veins between E8 and E9.5 needs VECadherin [63]. To form new vascular connections, tip cells at the angiogenic front need to suppress their motility upon encountering their targets, namely the tips of other sprouts or existing vessels [58]. Strong adhesive interactions and EC–EC junctional contacts mediated by vascular endothelial cadherin (VE-cadherin) need to be established at joining points [64]. VE-Cadherin is guided to the tips of EC filopodia by myosin X, a plus-end directed actin motor, thereby inducing early cell-cell contacts [65]. Blood vessels in the vascular plexus presumably have more stabilized adherence junctions than

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those of the angiogenic front. Consistent with this observation, ECspecific- inducible VE-cadherin KO mice show ectopical sprout formation even in the vascular plexus [66]. The process of EC tubule formation was recently visualized in living zebrafish using an EGFPtagged version of the junctional protein zona occludens 1 (ZO-1), a component of tight junctions [64]. Deletion of the ZO-1 gene in mice led to similar phenotypes and embryonic death as observed for the deletion of VE-Cadherin indicating that adherens junctions as well as tight junctions are necessary for tubule formation during development [67]. In adult organism, junctions have to be transiently disorganized e.g. for sprouting angiogenesis or diapedesis, or they must stay tight as in the blood-brain barrier. The stability of endothelial adherens junctions is mainly regulated by the plasma membrane localization of VE-Cadherin that is influenced by many factors ((Fig. 2) reviewed by [68]). The extracellular domain of VECadherin contains five cadherin-type repeats and binds to VECadherin molecules on neighboring endothelial cells. The proximal cytoplasmic part which regulates endocytosis binds to p120-catenin whereas the distal cytoplasmic tail binds to b-catenin or plakoglobin (g-catenin) which then binds to a-catenin and the actin cytoskeleton [69]. VEGFs (originally known as vascular permeability factors) and their receptors are the main factors that promote adherens junction disassembly in blood vessels, thus promoting vascular permeability [70]. Binding of VEGF-A to VEGFR2 leads to phosphorylation of tyrosines of VEGR2 including Y951. Phosphorylation of VEGFR2 at Y951 recruits the adapter protein TSAd which activates c-Src [71]. Activated c-Src induces phosphorylation of VE-Cadherin at multiple sites including Y658 which is localized in the binding site of p120catenin [72]. This phosphorylation leads to the dissociation of p120catenin from VE-Cadherin thereby unmasking an endocytic signal consisting of three amino acids (DEE) in the proximal cytoplasmic part of VE-Cadherin [73]. In addition, S665 is phorphorylated by a cSrc/ Wav2/Rac/PAK cascade leading to the recruitment of b-Arrestin2

VEGFR-2

p120 β-cat

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p120

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β-Arr2

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P p120

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VE-Cadherin DEP-1 and VE-PTP Occludin

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Fig. 2 – Modulation of the integrity of endothelial junction. VE-cadherin induces endothelial junctions with neighboring endothelial cells by forming a homotypic trans interactions. These junctions may also contain VEGFR2, phoshatases, and tight junction components like occludin. VEGF induces autophosphorylation of VEGFR2 that the leads (directly or indirectly) to phosphorylation of VE-cadherin and occludin. This can be counteracted by phosphatases, as long as the VEGFR2 concentration is low (left). If the VEGF concentration reaches a threshold, junction disassembly starts (right). Cytoplasmic components like catenins are released and become available for signaling processes. VEGFR2, VE-cadherin, and occludin are internalized. For VEGFR2 and VE-cadherin, it was shown that they internalize independently. It is unclear, if occludin traffics alone or together with VEGFR2 or VE-cadherin. Please cite this article as: M. Nakayama, P. Berger, Coordination of VEGF receptor trafficking and signaling by coreceptors, Exp Cell Res (2013), http://dx.doi.org/10.1016/j.yexcr.2013.03.008

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which is necessary for clathrin-based internalization of VE-Cadherin [74]. Internalization of VE-Cadherin and its subsequent degradation weakens adherens junctions. Many studies revealed that VECadherin and VEGFR2 coimmunprecipitate indicating that they cluster together in adherens junctions [63,74]. Interestingly, no colocalization of VEGFR2 and VE-Cadherin was found on internalized vesicles indicating that they are sorted out prior to endocytosis and that they have independent fates after internalization [52]. The weakening function of VEGFR2 at adherens junctions is counteracted by phosphatases that either dephosphorylate VEGFR2, VE-Cadherin or other components of cell junctions. Two receptor-type phosphatases, density-enhanced phosphatase-1 (DEP-1/CD148) and vascular endothelial protein tyrosine phosphatase (VE-PTP) constitutively colocalize with VEGFR2 and VE-cadherin in adherens junctions. DEP-1 associates with VECadherin through p120-catenin and b-catenin [52]. The resulting constitutive dephosphorylation of VEGFR2 generates a threshold preventing accidental activation of VEGFR2 and weakening of adherens junctions by minimal concentrations of VEGF in the blood. In addition, occludin and ZO-1 are dephosphorylated by DEP-1 suggesting that DEP-1 plays a role in maintaining tight junction integrity [75]. VE-PTP is the second phosphatase that associates with adherens junctions and it dephosphorylates VECadherin, VEGFR2, and VE-Cadherin. Binding of neutrophils and lymphocytes to endothelial cells leads to rapid dissociation of the VE-PTP/ VE-cadherin complex, followed by increased VEcadherin phosphorylation and destabilization of the junction indicating that VE-PTP plays a major role in regulating vascular permeability and leukocyte extravasation [76]. Adherens and tight junctions do not only colocalize at cell-cell junctions, they are also functionally linked. Transcription of claudin-5, a major component of the blood-brain barrier is regulated by adherens junctions. Plasma membrane bound VE-cadherin sequesters b-catenin from the cytoplasm, preventing the formation of the FoxO1/ b-catenin complex that blocks the transcription of claudin-5 [77]. This suggests that the formation of adherens junctions occurs prior to the formation of tight junctions. Vice versa, the degradation of VE-Cadherin leads to release of b-catenin preventing claudin-5 transcription. In addition, activated VEGFR2 leads to the phosphorylation and ubiquitination of occludin followed by internalization and transfer to late endosomes indicating that VEGFR2 also influences tight junction formation directly [78]. Under resting conditions, occludin and other tight junction components are constitutively internalized and recycled back to the plasma membrane in a Rab13 dependent process [79]. In a recent study, this Rab13-dependent process was linked to activated VEGFR2 and endothelial cell migration and angiogenesis [80].

Concluding remarks As we summarized here, previous reports have revealed that many key events of VEGFR signaling are controlled by intracellular receptor trafficking. After endocytosis, VEGFR is targeted through a recycling compartment back to the cell surface or to degradation. Although VEGF signal transduction can regulate a large variety of different cellular responses of endothelium, specific responses to VEGFs in a certain tissue will predominate in a context-dependent fashion. One possible hypothesis is tissue specific function of VEGF is linked to VEGF receptor trafficking

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regulated by other factors including the effect of coreceptors and cell-to-cell junction formation. To address this question, further analysis will be necessary.

Acknowledgments We thank Dr. Kurt Ballmer-Hofer for critical reading of the manuscript and Dr. Ralf Adams for the constructive discussion to write this review. Our work is supported by The Swiss National Science Foundation (Grant 31003A-118351 to P.B.), the ‘‘Novartis Stiftung fu¨r medizinisch-biologische Forschung’’ (to P.B.), the Max Planck Society (to M.N.), and The Japan Society for the Promotion of Science (to M.N.).

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