Necl2/3-mediated mechanism for tripartite synapse formation.

Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner, and synaptically-released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl2 trans-interacted with axonal Necl3, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl2, synaptically-released glutamate and axonal Necl3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl3 knockout and Necl2/3 double knockout mice confirmed these previously unreported mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.

s-Afadin binds to MAGUIN/Cnksr2 and regulates the localization of the AMPA receptor and glutamatergic synaptic response in hippocampal neurons.

A hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. Afadin has two splice variants: l-afadin and s-afadin. l-Afadin, but not s-afadin, regulates the formation of the PAJs but the roles of s-afadin in synaptogenesis remain unknown. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin in vivo and in vitro. MAGUIN/CNKSR2 is one of the causative genes for nonsyndromic X-linked intellectual disability accompanied by epilepsy and aphasia. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABAA receptor antagonist. These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model.

Identification of lysophosphatidic acid in serum as a factor that promotes epithelial apical junctional complex organization.

The apical junctional complex (AJC) consists of adherens junctions (AJs) and tight junctions and regulates epithelial integrity and remodeling. However, it is unclear how AJC organization is regulated based on environmental cues. We found here using cultured EpH4 mouse mammary epithelial cells that fetal bovine serum (FBS) in a culture medium showed an activity to promote AJC organization and that FBS showed an activity to promote tight junction formation even in the absence of AJ proteins, such as E-cadherin, αE-catenin, and afadin. Furthermore, we purified the individual factor responsible for these functions from FBS and identified this molecule as lysophosphatidic acid (LPA). In validation experiments, purified LPA elicited the same activity as FBS. In addition, we found that the AJC organization-promoting activity of LPA was mediated through the LPA receptor 1/5 via diacylglycerol-novel PKC and Rho-ROCK pathway activation in a mutually independent, but complementary, manner. We demonstrated that the Rho-ROCK pathway activation-mediated AJC organization was independent of myosin II-induced actomyosin contraction, although this signaling pathway was previously shown to induce myosin II activation. These findings are in contrast to the literature, as previous results suggested an AJC organization-disrupting activity of LPA. The present results indicate that LPA in serum has an AJC organization-promoting activity in a manner dependent on or independent of AJ proteins.

Nectin-2 in general and in the brain.

Nectins are immunoglobulin-like cell adhesion molecules constituting a family with four members, nectin-1, nectin-2, nectin-3, and nectin-4. In the brain, nectin-2 as well as nectin-1 and nectin-3 are expressed whereas nectin-4 is hardly expressed. In the nervous system, physiological functions of nectin-1 and nectin-3, such as synapse formation, mossy fiber trajectory regulation, interneurite affinity, contextual fear memory formation, and stress-related mental disorders, have been revealed. Nectin-2 is ubiquitously expressed in non-neuronal tissues and various nectin-2 functions in non-nervous systems have been extensively investigated, but nectin-2 functions in the brain have not been revealed until recently. Recent findings have revealed that nectin-2 is expressed in the specific areas of the brain and plays important roles, such as homeostasis of astrocytes and neurons and the formation of synapses. Moreover, a single nucleotide polymorphism in the human NECTIN2 gene is associated with Alzheimer's disease. We here summarize recent progress in our understanding of nectin-2 functions in the brain.

Nectins and Nectin-like molecules in synapse formation and involvement in neurological diseases.

Synapses are interneuronal junctions which form neuronal networks and play roles in a variety of functions, including learning and memory. Two types of junctions, synaptic junctions (SJs) and puncta adherentia junctions (PAJs), have been identified. SJs are found at all excitatory and inhibitory synapses whereas PAJs are found at excitatory synapses, but not inhibitory synapses, and particularly well developed at hippocampal mossy fiber giant excitatory synapses. Both SJs and PAJs are mediated by cell adhesion molecules (CAMs). Major CAMs at SJs are neuroligins-neurexins and Nectin-like molecules (Necls)/CADMs/SynCAMs whereas those at PAJs are nectins and cadherins. In addition to synaptic PAJs, extrasynaptic PAJs have been identified at contact sites between neighboring dendrites near synapses and regulate synapse formation. In addition to SJs and PAJs, a new type of cell adhesion apparatus different from these junctional apparatuses has been identified and named nectin/Necl spots. One nectin spot at contact sites between neighboring dendrites at extrasynaptic regions near synapses regulates synapse formation. Several members of nectins and Necls had been identified as viral receptors before finding their physiological functions as CAMs and evidence is accumulating that many nectins and Necls are related to onset and progression of neurological diseases. We review here nectin and Necls in synapse formation and involvement in neurological diseases.

Interaction of nectin-2α with the auxiliary protein of the voltage-gated A-type K+ channel Kv4.2 dipeptidyl aminopeptidase-like protein at the boundary between the adjacent somata of clustered cholinergic neurons in the medial habenula.

The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.

Requirement of the F-actin-binding activity of l-afadin for enhancing the formation of adherens and tight junctions.

The apical junctional complex consists of adherens junctions (AJs) and tight junctions (TJs) in polarized epithelial cells, which are attached to each other to form a sheet. Actin filaments (F-actin) are associated with AJs and TJs and required for the formation and maintenance of this complex. l-Afadin is an F-actin-binding protein, which is localized at AJs through binding to the cell adhesion molecule nectin, and regulates the formation of AJs and TJs. However, the role of the F-actin-binding activity of l-afadin for the formation of the apical junctional complex remains unknown. We generated here the cultured EpH4 mouse mammary epithelial cells in which afadin was genetically ablated. In the Ca2+ switch assay, the formation of both AJs and TJs was markedly impaired in the afadin-deficient cells. Re-expression of l-afadin in the afadin-deficient cells fully restored the formation of both AJs and TJs, but the re-expression of the l-afadin mutant lacking the FAB domain did not completely restore the formation of AJs or TJs. These results indicate that the F-actin-binding activity of l-afadin is required for enhancing the formation of both AJs and TJs.

Afadin Facilitates Vascular Endothelial Growth Factor-Induced Network Formation and Migration of Vascular Endothelial Cells by Inactivating Rho-Associated Kinase Through ArhGAP29.

OBJECTIVE: We previously reported that afadin, an actin filament-binding protein, regulated vascular endothelial growth factor-induced angiogenesis. However, the underlying molecular mechanisms are poorly understood. Here, we investigated the mechanisms of how Rho-associated kinase is activated in afadin-knockdown human umbilical vein endothelial cells (HUVECs) and how its activation is involved in defects of vascular endothelial growth factor-induced network formation and migration of the cells. APPROACH AND RESULTS: Knockdown of afadin or ArhGAP29, a GTPase-activating protein for RhoA, increased Rho-associated kinase activity and reduced the vascular endothelial growth factor-induced network formation and migration of cultured HUVECs, accompanied by the defective formation of membrane protrusions, such as lamellipodia and peripheral ruffles. Treatment of the afadin- or ArhGAP29-knockdown HUVECs with Rho-associated kinase inhibitors, Y-27632 or fasudil, partially restored the reduced network formation and migration as well as the defective formation of membrane protrusions. ArhGAP29 bound to afadin and was colocalized with afadin at the leading edge of migrating HUVECs. The defective formation of membrane protrusions in ArhGAP29-knockdown HUVECs was restored by expression of mutant ArhGAP29 that bound to afadin and contained a RhoGAP domain but not mutant ArhGAP29 that could bind to afadin and lacked the RhoGAP domain or mutant ArhGAP29 that could not bind to afadin and contained the RhoGAP domain. This suggested the requirement of both the interaction of afadin with ArhGAP29 and RhoGAP activity of ArhGAP29 for migration of HUVECs. CONCLUSIONS: Our results highlight a critical role of the afadin-ArhGAP29 axis for the regulation of Rho-associated kinase activity during vascular endothelial growth factor-induced network formation and migration of HUVECs.

Involvement of l-afadin, but not s-afadin, in the formation of puncta adherentia junctions of hippocampal synapses.

A hippocampal mossy fiber synapse has a complex structure in which presynaptic boutons attach to the dendritic trunk by puncta adherentia junctions (PAJs) and wrap multiply-branched spines, forming synaptic junctions. It was previously shown that afadin regulates the formation of the PAJs cooperatively with nectin-1, nectin-3, and N-cadherin. Afadin is a nectin-binding protein with two splice variants, l-afadin and s-afadin: l-afadin has an actin filament-binding domain, whereas s-afadin lacks it. It remains unknown which variant is involved in the formation of the PAJs or how afadin regulates it. We showed here that re-expression of l-afadin, but not s-afadin, in the afadin-deficient cultured hippocampal neurons in which the PAJ-like structure was disrupted, restored this structure as estimated by the accumulation of N-cadherin and αΝ-catenin. The l-afadin mutant, in which the actin filament-binding domain was deleted, or the l-afadin mutant, in which the αΝ-catenin-binding domain was deleted, did not restore the PAJ-like structure. These results indicate that l-afadin, but not s-afadin, regulates the formation of the hippocampal synapse PAJ-like structure through the binding to actin filaments and αN-catenin. We further found here that l-afadin bound αN-catenin, but not γ-catenin, whereas s-afadin bound γ-catenin, but hardly αN-catenin. These results suggest that the inability of s-afadin to form the hippocampal synapse PAJ-like structure is due to its inability to efficiently bind αN-catenin.

Aging-dependent expression of synapse-related proteins in the mouse brain.

A synapse is a cell adhesion structure that permits a neuron to pass a chemical or electrical signal to another neuron. They connect neurons and form neural networks that are essential for brain functions, such as learning and memory. At a chemical synapse, the presynapse and the postsynapse are connected by cell adhesion molecules. The presynapse contains synaptic vesicles and their release machinery, whereas the postsynapse contains postsynaptic densities and receptors for the neurotransmitters. Many proteins constituting a synapse have been identified, but their life-span expression profiles remain elusive. Here, we investigated the expression levels of representative synapse-related proteins by Western blot using the extranuclear supernatant fraction of the brains of mice at various ages. These proteins were classified into seven groups depending on their expression profiles during the embryonic stage, those from postnatal day 6 (P6) to P30, and those after P90. The expression levels of the majority of the proteins were gradually increased from the embryonic stage and then decreased at P14 or P30. After P90, the expression levels were not markedly changed or, in some proteins, increased. These results indicate that the expression levels of the synapse-related proteins are regulated orderly in an aging-dependent manner.

Roles of afadin in functional differentiations of hippocampal mossy fiber synapse.

A hippocampal mossy fiber synapse has a complex structure and is implicated in learning and memory. In this synapse, the mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions and wrap around a multiply-branched spine, forming synaptic junctions. We have recently shown using transmission electron microscopy, immunoelectron microscopy and serial block face-scanning electron microscopy that atypical puncta adherentia junctions are formed in the afadin-deficient mossy fiber synapse and that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities and the density of synaptic vesicles docked to active zones are decreased in the afadin-deficient synapse. We investigated here the roles of afadin in the functional differentiations of the mossy fiber synapse using the afadin-deficient mice. The electrophysiological studies showed that both the release probability of glutamate and the postsynaptic responsiveness to glutamate were markedly reduced, but not completely lost, in the afadin-deficient mossy fiber synapse, whereas neither long-term potentiation nor long-term depression was affected. These results indicate that afadin plays roles in the functional differentiations of the presynapse and the postsynapse of the hippocampal mossy fiber synapse.

NGL-3-induced presynaptic differentiation of hippocampal neurons in an afadin-dependent, nectin-1-independent manner.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure. We have previously shown using afadin-deficient mice that afadin plays multiple roles in the structural and functional differentiations of this synapse. We investigated here using a co-culture system with cultured hippocampal neurons and non-neuronal COS-7 cells expressing synaptogenic cell adhesion molecules (CAMs) whether afadin is involved in the presynaptic differentiation of hippocampal synapses. Postsynaptic CAMs NGL-3 (alias, a Lrrc4b gene product) and neuroligin induced presynaptic differentiation by trans-interacting with their respective presynaptic binding CAMs LAR (alias, a Ptprf gene product) and neurexin. This activity of NGL-3, but not neuroligin, was dependent on afadin, but not the afadin-binding presynaptic CAM nectin-1. The afadin-binding postsynaptic CAM nectin-3 did not induce presynaptic differentiation. Immunofluorescence and immunoelectron microscopy analyses showed that afadin was localized mainly at puncta adherentia junctions, but partly at synaptic junctions, of the mossy fiber synapse. ß-Catenin and γ-catenin known to bind to LAR were co-immunoprecipitated with afadin from the lysate of mouse brain. These results suggest that afadin is involved in the NGL-3-LAR system-induced presynaptic differentiation of hippocampal neurons cooperatively with ß-catenin and γ-catenin in a nectin-1-independent manner.

Roles of afadin in the formation of the cellular architecture of the mouse hippocampus and dentate gyrus.

The hippocampal formation with tightly packed neurons, mainly at the dentate gyrus, CA3, CA2, and CA1 regions, constitutes a one-way neural circuit, which is associated with learning and memory. We previously showed that the cell adhesion molecules nectins and its binding protein afadin play roles in the formation of the mossy fiber synapses which are formed between the mossy fibers of the dentate gyrus granule cells and the dendrites of the CA3 pyramidal cells. We showed here that in the afadin-deficient hippocampal formation, the dentate gyrus granules cells and the CA3, CA2, and CA1 pyramidal cells were abnormally located; the mossy fiber trajectory was abnormally elongated; the CA3 pyramidal cells were abnormally differentiated; and the densities of the presynaptic boutons on the mossy fibers and the apical dendrites of the CA3 pyramidal cells were decreased. These results indicate that afadin plays roles not only in the formation of the mossy fiber synapses but also in the formation of the cellular architecture of the hippocampus and the dentate gyrus.

s-Afadin binds more preferentially to the cell adhesion molecules nectins than l-afadin.

l-Afadin was originally purified from rat brain as an actin filament (F-actin)-binding protein that was homologous to the AF-6 gene product. Concomitantly, s-afadin that did not show an F-actin-binding capability was copurified with l-afadin. Structurally, s-afadin lacks the C-terminal F-actin-binding domain but has two short sequences that were not present in l-afadin. The properties and roles of l-afadin have intensively been investigated, but those of s-afadin have poorly been understood. We show here an additional difference in their biochemical properties other than binding to F-actin between l-afadin and s-afadin. Both l-afadin and s-afadin bound to nectins, immunoglobulin-like cell adhesion molecules, whereas s-afadin more preferentially bound to nectins than l-afadin. The PDZ domain of l-afadin and s-afadin was essential for their binding to nectin-3. The dilute domain of l-afadin negatively regulated its binding to nectin-3, but the deletion of the C-terminal F-actin-binding domain of l-afadin did not increase the binding of l-afadin to nectin-3. These results indicate that the s-afadin-specific C-terminal inserts may be involved in its preference of binding to nectin-3 and raise the possibility that there are proteins other than nectins that more preferentially bind s-afadin than l-afadin.

Necl-5/poliovirus receptor interacts with VEGFR2 and regulates VEGF-induced angiogenesis.

RATIONALE: Vascular endothelial growth factor (VEGF), a major proangiogenic agent, exerts its proangiogenic action by binding to VEGF receptor 2 (VEGFR2), the activity of which is regulated by direct interactions with other cell surface proteins, including integrin α(V)ß(3). However, how the interaction between VEGFR2 and integrin α(V)ß(3) is regulated is not clear. OBJECTIVE: To investigate whether Necl-5/poliovirus receptor, an immunoglobulin-like molecule that is known to bind integrin α(V)ß(3), regulates the interaction between VEGFR2 and integrin α(V)ß(3), and to clarify the role of Necl-5 in the VEGF-induced angiogenesis. METHODS AND RESULTS: Necl-5-knockout mice displayed no obvious defect in vascular development; however, recovery of blood flow after hindlimb ischemia and the VEGF-induced neovascularization in implanted Matrigel plugs were impaired in Necl-5-knockout mice. To clarify the mechanism of the regulation of angiogenesis by Necl-5, we investigated the roles of Necl-5 in the VEGF-induced angiogenic responses in vitro. Knockdown of Necl-5 by siRNAs in human umbilical vein endothelial cells (HUVECs) inhibited the VEGF-induced capillary-like network formation on Matrigel, migration, and proliferation, and conversely, enhanced apoptosis. Coimmunoprecipitation assays showed the interaction of Necl-5 with VEGFR2, and knockdown of Necl-5 prevented the VEGF-induced interaction of integrin α(V)ß(3) with VEGFR2. Knockdown of Necl-5 suppressed the VEGFR2-mediated activation of downstream proangiogenic and survival signals, including Rap1, Akt, and endothelial nitric oxide synthase. CONCLUSIONS: These results demonstrate the critical role of Necl-5 in angiogenesis and suggest that Necl-5 may regulate the VEGF-induced angiogenesis by controlling the interaction of VEGFR2 with integrin α(v)ß(3), and the VEGFR2-mediated Rap1-Akt signaling pathway.

FGD5 mediates proangiogenic action of vascular endothelial growth factor in human vascular endothelial cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF) exerts proangiogenic action and induces activation of a variety of proangiogenic signaling pathways, including the Rho family small G proteins. However, regulators of the Rho family small G proteins in vascular endothelial cells (ECs) are poorly understood. Here we attempted to clarify the expression, subcellular localization, downstream effectors, and proangiogenic role of FGD5, a member of the FGD family of guanine nucleotide exchange factors. METHODS AND RESULTS: FGD5 was shown to be selectively expressed in cultured human vascular ECs. Immunofluorescence microscopy showed that the signal for FGD5 was observed at peripheral membrane ruffles and perinuclear regions in human umbilical vein ECs. Overexpression of FGD5 increased Cdc42 activity, whereas knockdown of FGD5 by small interfering RNAs inhibited the VEGF-induced activation of Cdc42 and extracellular signal-regulated kinase. VEGF-promoted capillary-like network formation, permeability, directional movement, and proliferation of human umbilical vein ECs and the reorientation of the Golgi complex during directional cell movement were attenuated by knockdown of FGD5. CONCLUSIONS: This study provides the first demonstration of expression, subcellular localization, and function of FGD5 in vascular ECs. The results suggest that FGD5 regulates proangiogenic action of VEGF in vascular ECs, including network formation, permeability, directional movement, and proliferation.

Heterogeneity of perivascular astrocyte endfeet depending on vascular regions in the mouse brain.

Astrocytes interact with not only synapses but also brain blood vessels through perivascular astrocyte endfeet (PV-AEF) to form the neurovascular unit (NVU). However, PV-AEF components have not been fully identified. Here, we biochemically isolated blood vessels from mouse brain homogenates and purified PV-AEF. The purified PV-AEF were observed in different sizes, similar to PV-AEF on brain blood vessels. Mass spectrometry analysis identified 9,762 proteins in the purified PV-AEF, including cell adhesion molecules, nectin-2δ, Kirrel2, and podoplanin. Immunofluorescence microscopic analysis revealed that nectin-2δ and podoplanin were concentrated mainly in arteries/arterioles and veins/venules of the mouse brain, whereas Kirrel2 was mainly in arteries/arterioles. Nectin-2α/δ, Kirrel2, and podoplanin were preferentially observed in large sizes of the purified PV-AEF. Furthermore, Kirrel2 potentially has cell adhesion activity of cultured astrocytes. Collectively, these results indicate that PV-AEF have heterogeneity in sizes and molecular components, implying different roles of PV-AEF in NVU function depending on vascular regions.

Role of afadin in vascular endothelial growth factor- and sphingosine 1-phosphate-induced angiogenesis.

RATIONALE: Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis. OBJECTIVE: We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism. METHODS AND RESULTS: Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell-cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin-PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K-Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis. CONCLUSIONS: These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.

Involvement of the interaction of afadin with ZO-1 in the formation of tight junctions in Madin-Darby canine kidney cells.

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with beta- and alpha-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-DeltaPR1-2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.

Localization of nectin-free afadin at the leading edge and its involvement in directional cell movement induced by platelet-derived growth factor.

Afadin is an actin-filament-binding protein that binds to nectin, an immunoglobulin-like cell-cell adhesion molecule, and plays an important role in the formation of adherens junctions. Here, we show that afadin, which did not bind to nectin and was localized at the leading edge of moving cells, has another role: enhancement of the directional, but not random, cell movement. When NIH3T3 cells were stimulated with platelet-derived growth factor (PDGF), afadin colocalized with PDGF receptor, alphavbeta3 integrin and nectin-like molecule-5 at the leading edge and facilitated the formation of leading-edge structures and directional cell movement in the direction of PDGF stimulation. However, these phenotypes were markedly perturbed by knockdown of afadin, and were dependent on the binding of afadin to active Rap1. Binding of Rap1 to afadin was necessary for the recruitment of afadin and the tyrosine phosphatase SHP-2 to the leading edge. SHP-2 was previously reported to tightly regulate the activation of PDGF receptor and its downstream signaling pathway for the formation of the leading edge. These results indicate that afadin has a novel role in PDGF-induced directional cell movement, presumably in cooperation with active Rap1 and SHP-2.