Neuropilin-1/GIPC1 Signaling Regulates α5β1 Integrin Traffic and Function in Endothelial Cells

1In vertebrates, the development of a hierarchically organized and functional vascular tree relies on the dynamic interaction of endothelial cells (ECs) with the surrounding extracellular matrix (ECM), which is mediated by heterodimeric αβ integrin adhesive receptors. During evolution, vertebrates have acquired an additional set of adhesion-related genes that regulate blood vessel assembly and function. Among these genes, the ECM protein fibronectin (FN) and α5β1 integrin, the predominant FN receptor, have proven to be essential for embryonic vascular development and tumor angiogenesis. Indeed, in vertebrate embryos FN is the earliest and most abundantly expressed subendothelial matrix molecule. Endothelial α5β1 mediates cell adhesion to FN and the assembly of soluble FN dimers (sFN) into a fibrillar network, which has also been implicated in branching morphogenesis.

2The biological activities of integrins depend on the dynamic regulation of their adhesive function in space and time. In cells, integrins exist in different conformations that determine their affinities for ECM proteins and are continuously endocytosed, trafficked through endosomal compartments, and recycled back to the plasma membrane. Therefore, during vascular morphogenesis, real-time modulation of EC–ECM adhesion can result from two interconnected phenomena: the regulation of integrin conformation and traffic in response to extracellular stimuli. Indeed, there is mounting evidence that pro- and antiangiogenic cues regulate blood vessel formation by modulating integrin function. In this respect, the transmembrane glycoprotein neuropilin 1 (Nrp1), which is expressed in both neurons and ECs, is remarkable because it was originally identified as a surface protein mediating cell adhesion and then found to also act as a coreceptor for both pro- and antiangiogenic factors, such as vascular endothelial growth factor A 165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A), respectively.

3The extracellular region of Nrp1 contains two repeated complement-binding domains (CUB domains; a1-a2 domains), two coagulation-factor-like domains (b1-b2 domains), and a juxtamembrane meprin/A5/μ-phosphatase (MAM; c) homology domain. The Nrp1 intracellular region is only 50 amino acids in length, and its function is poorly characterized. Through its b1-b2 domains, Nrp1 binds and potentiates the proangiogenic activity of VEGF-A165, which contains the heparin-binding peptide encoded by exon 7. In addition, Nrp1 acts as the ligand-binding subunit of the receptor complex for the antiangiogenic SEMA3A, whose sema and immunoglobulin-basic domains, respectively, bind the a1-a2 and b1-b2 domains of Nrp1. The MAM/c domain instead mediates the SEMA3A-elicited Nrp1 oligomerization that is required for SEMA3A biological activity. Interestingly, the short cytoplasmic domain of Nrp1 is not required for SEMA3A signaling in neurons. In addition, the extracellular b1-b2 domains of Nrp1 mediate heterophilic cell adhesion independently of VEGF-A165 and SEMA3A.

4Nrp1 null mice display an embryonic lethal phenotype, characterized by dramatic vascular defects ascribed to impaired angiogenic sprouting, branching, or arterialization that is significantly more severe than and/or qualitatively different from that of mice lacking either VEGF-A164 (VEGF120/120 mice) or Sema3A. Indeed, although Nrp1 –/– embryos die in utero by 13.5 days postcopulation, VEGF120/120 pups are recovered at birth at a normal Mendelian frequency. Moreover, a major feature of Nrp1 null mutants, i.e., the severe impairment of neural tube vascularization, is not phenocopied by VEGF120/120 mouse embryos. In addition, differently from Nrp1 null mice, the vascular phenotype of Sema3a null mice is significantly influenced by the genetic background. These findings suggest that the vascular defects caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in vascular cells, such as adhesion to the ECM. However, how Nrp1 regulates integrin-dependent EC linkages to the surrounding matrix is still obscure. Here, we shed light on the molecular mechanisms by which Nrp1, via its short cytoplasmic domain and independently of VEGF-A165 and SEMA3A, specifically controls a biological function that is crucial for vascular development, namely, α5β1-mediated EC adhesion to FN.

5To understand the mechanisms by which Nrp1 modulates EC adhesion to different ECM proteins, we silenced the expression of Nrp1 in human umbilical artery ECs by RNA interference (RNAi). Parenthetically, Nrp1 has been found to be expressed at higher levels in arteries than in veins. Endothelial cells were transfected twice with either a pool of three different small interfering RNAs (siRNAs) targeting human Nrp1 (sihNrp1) or control nontargeting siRNA (siCtl). Twenty-four hours after the second transfection, Western blot analysis revealed that, in comparison with control cells, Nrp1 protein, but neither β-tubulin nor the Nrp1 interactor GAIP interacting protein C terminus, member 1 (GIPC1), was successfully silenced in sihNrp1 ECs (Figure 1A).

6Next, we investigated the effect of Nrp1 silencing on EC adhesion to different ECM proteins. Fibronectin, vitronectin (VN), and type I collagen (COLL-I) are typical constituents of the provisional angiogenic ECM, whereas laminin (LN) isoforms are major components of the vascular basement membrane surrounding both immature and mature blood vessels. Short-term (15 min) adhesion assays showed that loss of Nrp1 greatly reduced EC adhesion to FN but not to VN, COLL-I, or LN (Figure 1B–E), suggesting that positive modulation of cell adhesion by Nrp1 is not a general phenomenon but rather a function restricted to specific ECM proteins, such as FN.

7Because FN polymerization by ECs has been suggested to participate in vascular morphogenesis, we next examined the role of Nrp1 in the fibrillogenesis of endogenous FN. During FN matrix assembly, current models envisage the binding of sFN to surface integrins, thus causing the conversion of FN to a conformational form that favors fibril formation through interactions with other integrin-bound FN dimers. Endothelial cells were cultured in a medium containing FN-depleted fetal calf serum, and accumulation of endogenous FN into fibrils was then detected by confocal immunofluorescence analysis. In comparison with control cells, sihNrp1 ECs were impaired in their ability to incorporate endogenous sFN into a dense fibrillar network 3 h after plating (Figure 1F and 1G). Time-course real-time reverse transcription PCR (RT-PCR) and Western blot analyses revealed that the endogenous FN fibrillogenesis defect observed in sihNrp1 ECs was not due to a reduction in FN mRNA (Figure S1A) or protein (Figure S1D). Hence, Nrp1 specifically promotes EC adhesion to FN and FN matrix formation.

8To start dissecting the mechanisms by which Nrp1 controls the interaction of human ECs with FN, we sought to compare the abilities of full-length and deletion constructs of mouse Nrp1 (mNrp1) to rescue the adhesion and fibrillogenesis defects of sihNrp1 ECs (Figure 2). In particular, we investigated the role played by the extracellular and cytoplasmic moieties of Nrp1. Indeed, the Nrp1 cytodomain, although dispensable for SEMA3A collapsing activity in neurons, could signal in cultured ECs. Moreover, the C-terminal SEA sequence of Nrp1 interacts with the PDZ domain of the endocytic adaptor protein GIPC1, whose knockdown during development results in altered arterial branching. Therefore, we transduced sihNrp1 ECs with retroviral vectors carrying the hemagglutinin (HA)-tagged full-length (mNrp1) and deletion mutants of murine Nrp1 (Figure 2A), lacking either the C-terminal SEA amino acids (mNrp1dSEA) or the whole cytoplasmic domain (mNrp1dCy). The sihNrp1 pool did not target any of the mNrp1 constructs, and immunoprecipitation experiments on membrane-biotinylated cell monolayers revealed that all three transmembrane proteins were efficiently exposed on the cell surface (Figure 2B).

9In comparison to wild-type mNrp1, both mNrp1dSEA and mNrp1dCy constructs were severely impaired in their abilities to rescue sihNrp1 EC defects in adhesion to FN (Figure 2C) and endogenous FN fibrillogenesis (Figure 2D–F). Accordingly, only mNrp1 overexpression stimulated the adhesion of NIH 3T3 fibroblasts to FN, whereas neither mNrp1dSEA nor mNrp1dCy were active in this respect (Figure 2G). Moreover, mNrp1 overexpression did not promote NIH 3T3 adhesion to VN (Figure S2), further supporting the concept that Nrp1 behaves as a substrate-specific enhancer of cell adhesion. Hence, it appears that the cytoplasmic domain of Nrp1, in particular its SEA motif, which interacts with the endocytic adaptor GIPC1, is required for Nrp1 stimulation of EC spreading on FN and polymerization of endogenous FN.

10Opposing autocrine loops of VEGF-A and SEMA3A have been found in ECs both in vitro and in vivo. Therefore, we investigated whether the SEA motif and the full cytoplasmic domain of Nrp1 could be required for the modulation of EC adhesion to FN by VEGF-A165 and SEMA3A. Consistent with previous observations, silencing Nrp1 completely blocked VEGF-A165-dependent stimulation (Figure 2H) and SEMA3A-dependent inhibition (Figure 2I) of human EC adhesion to FN. As expected, inhibition of cell adhesion to FN by SEMA3F, which signals through Nrp2, was not affected by Nrp1 knockdown (Figure 2J). Moreover, similarly to what was observed for SEMA3A in neurons, we found that the cytoplasmic domain of Nrp1 is entirely dispensable for both VEGF-A165 (Figure 2H) and SEMA3A (Figure 2I) activity on EC adhesion to FN, because all three mNrp1 constructs rescued sihNrp1 EC response to these factors with a similar efficiency. Thus, the Nrp1 SEA motif and cytodomain are required for Nrp1 modulation of EC adhesion to FN and sFN incorporation into fibrils but not for Nrp1 activity as a VEGF-A165 and SEMA3A coreceptor.

11α5β1 Integrin is the main FN receptor in ECs, and by transmitting the actin-dependent tension to sFN, it triggers FN fibrillogenesis. To elucidate whether Nrp1 stimulation of cell adhesion to FN was directly mediated by Nrp1 or was dependent on α5β1 integrin, CHO cells lacking (CHO B2) or expressing (CHO B2α27) the α5 integrin subunit were transfected with mNrp1 and allowed to adhere to FN. Overexpression of mNrp1 stimulated CHO cell adhesion to FN in the presence (CHO B2α27; Figure 3A) but not in the absence (CHO B2; Figure 3B) of α5β1 integrin. Therefore, Nrp1′s proadhesive activity on FN is nonautonomous and mediated by α5β1 integrin.

12We then examined whether in ECs Nrp1 could interact physically with α5β1 integrin. Lysates from ECs adhering on endogenous ECM were immunoprecipitated with an antibody (Ab) recognizing the FN receptor α5β1 and then blotted with anti-Nrp1 Ab. Nrp1 coimmunoprecipitated with α5β1, and blotting Nrp1 immunoprecipitates with anti-α5β1-integrin Ab further confirmed the association between endogenous hNrp1 and α5β1 integrin in ECs (Figure 3C). To better understand whether the Nrp1 cytoplasmic domain was required for the interaction with α5β1 integrin, lysates of NIH 3T3 fibroblasts overexpressing HA-tagged full-length or deletion constructs of mNrp1 and green fluorescent protein (GFP)-tagged α5 integrin subunit (α5-GFP) were immunoprecipitated with anti-GFP Ab and then blotted with anti-HA Ab (Figure 3D). We found that both the C-terminal SEA and the cytoplasmic domain of Nrp1 were fully dispensable for its interaction with α5β1 integrin.

13To understand the spatial and functional relationships between Nrp1 and α5β1 integrin in ECs, we first generated a monomeric red fluorescent protein (mRFP)-tagged mNrp1 construct (mNrp1-mRFP) that was then cotransfected with α5-GFP in ECs. Fluorescent confocal microscopy showed that at the plasma membrane of ECs adhering on FN mNrp1-mRFP was enriched in close proximity to, or even tightly intermingled with, α5-GFP-containing adhesion sites (Figure 4A, arrows). Moreover, mNrp1-mRFP and α5-GFP fully colocalized in intracellular vesicles (Figure 4A, arrowheads). Notably, immunofluorescence analysis of endogenous endothelial proteins confirmed the spatial links between hNrp1 and vinculin (Figure 4B) or α5β1 integrin (Figure 4C) at either adhesion sites (Figure 4B and 4C, arrows) or vesicular structures located in their proximity (Figure 4C, arrowheads). The observation that Nrp1 and α5β1 colocalization was particularly apparent in intracellular vesicles indicated that these two molecules may associate at or near the point of endocytosis and that they may be internalized as a complex, which is then subsequently disassembled upon recycling to the plasma membrane. We have previously found that endosomal integrin complexes can be preserved by treating the cell with primaquine (PMQ), a receptor recycling inhibitor, prior to lysis. Therefore, we immunoprecipitated α5β1 integrin or Nrp1 from cells that had been treated with PMQ for 10 min and probed for the presence of the α5β1/Nrp1 complex by Western blotting. Pretreatment of the cells with PMQ greatly increased the coprecipitation of α5β1 integrin with Nrp1 and vice versa (Figure 3C), indicating the likelihood that this complex is more stable in endosomes than at the plasma membrane.

14To further characterize the interaction between Nrp1 and α5β1 integrin, we measured fluorescence resonance energy transfer (FRET) in live NIH 3T3 cells transfected with α5-GFP alone or cotransfected with α5-GFP and mNrp1 tagged with the fluorescent protein Cherry, an improved version of mRFP (mNrp1-Cherry). Total internal reflection fluorescence (TIRF) illumination was used to selectively excite α5-GFP at the basal cell plasma membrane where ECM adhesions lie. Fluorescence resonance energy transfer was measured by fluorescence lifetime imaging microscopy (FLIM) and was read out as a decrease in donor (GFP) fluorescence lifetime. We found that the α5-GFP fluorescence lifetime was significantly reduced in cells that coexpressed mNrp1-Cherry, indicating that FRET, and thus a close physical interaction, was occurring between α5β1 and Nrp1 at adhesion sites with an 11.5% FRET efficiency (Figure 5).

15Taken together, these data indicate that in living cells Nrp1 physically associates with α5β1 at or near sites of cell–ECM contact and that this interaction is likely maintained following internalization of the complex.

16The efficiency of cell adhesion and spreading on ECM is generally thought to be proportional to the amount of either active or total (i.e., active and inactive) integrin at the cell surface. We found that lack of Nrp1 did not alter the global amount of either total (Figure 1A), as already reported, or active α5β1 integrin, as recognized by the mouse monoclonal Ab (mAb) SNAKA51 (Figure S3). Then, we analyzed whether Nrp1 could influence the amount of α5β1 integrin on the endothelial surface. Biotinylation experiments revealed that knocking down human Nrp1 did not diminish the surface levels of either total or active α5β1 integrin in sihNrp1 ECs (Figure 6A), thus suggesting that a mechanism alternative to the control of integrin conformation should be responsible for Nrp1-dependent activation of α5β1 integrin function in ECs.

17On the basis of our observations that Nrp1 and α5β1 integrin colocalize in intracellular vesicles (Figure 4A and 4C) and that inhibition of recycling by PMQ increased the association of Nrp1 with α5β1 integrin (Figure 3C), we decided to monitor the effect of Nrp1 knockdown on the internalization of total and active surface α5β1 integrin. Endothelial cells were surface-labeled with cleavable biotin at 4 °C and incubated at 37 °C for different times to allow internalization, and then biotin remaining on cell-surface proteins was cleaved at 4 °C. Integrin internalization was quantified by immunoprecipitation of either total (Figure 6A and 6B) or active (Figure 6A and 6C) α5β1 integrin, followed by Western blot analysis with streptavidin. Notably, although endocytosis of the cell-surface pool of total α5β1 integrin (i.e., active plus inactive heterodimers) was not detectably altered in sihNrp1 cells (Figure 6A and 6B), knockdown of Nrp1 markedly reduced the quantity of active (SNAKA51-positive) α5β1 heterodimers internalized by ECs (Figure 6A and 6C). Taken together, these data indicate that on the cell surface Nrp1 interacts with active α5β1 heterodimers at adhesion sites (Figure 4A and 4C, arrows) and acts to promote their internalization and localization to intracellular vesicles (Figure 4A and 4C, arrowheads).

18To visualize the internalization and postendocytic trafficking of the α5β1/Nrp1 complex, we deployed the photoactivatable (PA) α5-GFP (α5-PA-GFP) probe that we had previously used to monitor α5β1 trafficking in human ovarian carcinoma A2780 cells. However, the multitude of fluorescent vesicles travelling to and from the cell surface made it difficult to track the progress of individual α5β1 integrin transport vesicles. Therefore, we used TIRF to restrict the plane of activating fluorescence, such that only α5-PA-GFP present at or near the cell surface became photoactivated. Then we tracked the movement of this photoactivated fraction of α5β1 integrin using time-lapse epifluorecence microscopy. With this novel technique, α5β1 integrin was photoactivated almost exclusively at adhesion sites (mostly fibrillar adhesions), where it colocalized with mNrp1-Cherry (Figure 7, arrows). Photoactivated α5β1 was then rapidly (<6 s) internalized and cotransported with mNrp1-Cherry in small endocytic vesicles (Figure 7, empty arrowheads, and Video S1) that moved away from the fibrillar adhesions. In addition, we found that α5β1 integrin turnover in ECM adhesions was unexpectedly very rapid (Figure S4 and Video S2), with the α5-PA-GFP signal leaving the adhesive sites, accumulating in vesicles, and disappearing by ∼45 s after photoactivation in approximately 50% of the adhesion sites and by ∼115 s in the remaining ones (Video S2).

19Having established that α5β1 integrin and Nrp1 are cointernalized at fibrillar adhesions, we wished to determine whether the integrin was then recycled from Nrp1-positive vesicles back to the plasma membrane. To address this, we aimed a pulse of 405-nm laser light at a “single point” corresponding to Nrp1-positive vesicles, leading to the immediate photoactivation of α5-PA-GFP integrin largely within the confines of these structures (Figure 8 and Video S3). During the following 80 s, fluorescence was lost from the photoactivated vesicle, and this was accompanied by a corresponding increase in integrin fluorescence at peripheral elongated structures that look like adhesion sites (Figure 8, red circle and white arrows). On the contrary, when the activating laser was aimed at a cell region devoid of Nrp1-positive vesicles, little or no photoactivation occurred (Figure S5 and Video S4), indicating that the α5-PA-GFP fluorescence detected in Figure 8 was indeed at mNrp1-Cherry vesicles and not at the plasma membrane above and below them.

20Taken together, these data indicate that α5β1 integrin and Nrp1 are cointernalized into intracellular vesicles, which are then rapidly returned or recycled to the plasma membrane. Interestingly, both the internalization and the recycling of Nrp1-associated α5β1 integrin occur at the site of adhesion to the ECM.

21In the eukaryotic early endocytic pathway, the small GTPase Rab5 is a rate-limiting component that regulates the entry of cargoes from the plasma membrane into the early endosome. Hence, we analyzed the early endocytic steps of active α5β1 integrin in ECs cotransfected with mNrp1-mRFP and Rab5-GFP, which were incubated with the α5β1 integrin activation reporter mAb SNAKA51 for 30 min at 4 °C and then at 37 °C for different time points. Fluorescent confocal analysis indicated that, after 1–3 min of internalization at 37 °C, Nrp1 and active α5β1 integrin colocalized in early Rab5-positive vesicles near the EC plasma membrane (Figure 9A). Accordingly, immunofluorescence analysis of endogenous endothelial proteins confirmed that hNrp1 and Rab5 colocalized in vesicles, many of which were located near adhesion sites (Figure 9B, empty arrowheads), further supporting the view that Nrp1 can induce α5β1-mediated adhesion by promoting the preferential internalization of its active conformation into Rab5-positive early endosomes and the ensuing recycling to newly forming cell–ECM contacts.

22Next, to characterize the molecular mechanisms by which Nrp1 regulates the traffic of active α5β1 integrin, we evaluated the abilities of mNrp1 full-length and mutant constructs to rescue the integrin internalization defects that we observed in sihNrp1 ECs. Remarkably, only wild-type mNrp1, but neither mNrp1dSEA nor mNrp1dCy construct, was able to rescue the sihNrp1 EC defects in the endocytosis of active α5β1 integrin (Figure 6D). Therefore, in ECs the SEA motif of Nrp1, which binds the endocytic adaptor GIPC1, is mandatory for Nrp1 stimulation of cell adhesion to FN (Figure 2C), endogenous FN fibrillogenesis (Figure 2D–F), and active α5β1 integrin endocytosis (Figure 6D).

23The N-terminal portion of GIPC1 mediates its oligomerization, whereas its central PDZ domain can bind the C-terminal consensus S/T-X-Φ sequence of Nrp1, the α5 integrin subunit, and the Rab5/Rab21 interactor protein APPL1. Thus, we theorized that as a result GIPC1 could support the Rab5-dependent early internalization of α5β1 integrin. To test this hypothesis, we silenced the expression of GIPC1 in human umbilical artery ECs by RNAi and examined its effect on α5β1 integrin traffic. Western blot analysis showed that, 96 h after the second transfection, GIPC1 protein, but not β-tubulin, was successfully silenced in sihGIPC1 ECs in comparison with control cells (Figure 10A). Knockdown of GIPC1 in ECs dramatically reduced the amount of internalized total (Figure 10C and 10D) and active (Figure 10C and 10E) α5β1 integrin by ∼70% throughout the whole internalization assay, suggesting that indeed the interaction of α5β1 integrin with GIPC1 is crucial for the endocytosis and the proper functioning of this integrin. Accordingly, short-term adhesion assays showed that, in comparison with control cells, sihGIPC1 ECs adhered poorly to FN (Figure 10B) and much less efficiently assembled endogenous sFN into a fibrillar network (Figure S1H) in comparison with cells transfected with siCtl (Figure S1G). The latter defect was not due to a reduction in FN mRNA or protein levels as demonstrated by real-time RT-PCR (Figure S1B) and Western blotting (Figure S1E). Hence, within Nrp1 the extracellular domain mediates the association with α5β1 integrin, and the C-terminal SEA sequence allows the binding to the endocytic adaptor GIPC1 that stimulates the internalization and traffic of active α5β1 integrin, finally promoting EC adhesion to FN and FN fibrillogenesis.

24Because the C terminus of GIPC1 binds to the minus-end-directed motor myosin VI (Myo6) that has also been involved in endocytosis, we considered the hypothesis that Myo6 could cooperate with GIPC1 in promoting α5β1 integrin internalization. Interestingly, RNAi-mediated knockdown of Myo6 in human umbilical artery ECs (Figure 10A) resulted in a significant (∼70%) impairment of active α5β1 integrin internalization (Figure 10F and 10H), whereas the total integrin pool was only mildly affected (∼25%; Figure 10F and 10G). These data, together with the fact that sihMyo6 EC adhesion to FN was severely hampered (Figure 10B), indicate that Myo6 cooperates with GIPC1 in the regulation of active α5β1 integrin endocytosis.

25Similarly to what we noticed after Nrp1 and GIPC1 knockdown, ECs in which Myo6 was silenced did not efficiently assemble an endogenous FN fibrillar network (Figure S1I) in comparison with cells transfected with siCtl (Figure S1G). However, differently from what we observed in sihNrp1 and sihGIPC1 ECs, the endogenous FN fibrillogenesis defect seen in sihMyo6 ECs was due to an inhibition of FN1 gene transcription mRNA (Figure S1C), which associated to a significant reduction of FN protein levels as well (Figure S1F). Indeed, in addition to its role in cytoplasmic transporting and anchoring, Myo6 is also present in the nucleus, where it promotes the RNA-polymerase-II-dependent transcription of active genes. Here we identify the FN1 gene as a new Myo6 transcriptional target and downstream effector that can bolster EC adhesion and motility.

26Defects of developing blood vessels caused by Nrp1 gene knockdown in mice are different from vascular malformations displayed by mice lacking either SEMA3A or VEGF-A165 (Vegf-a120/120 mice). Furthermore, it has been recently reported that Nrp1 is required for EC responses to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Therefore, it is conceivable that the vascular abnormalities of Nrp1–/– mice could be due at least in part to the disruption of a VEGF-A165/SEMA3A-independent Nrp1 function. α5β1 Integrin and its ligand FN are key players in vascular development. The data reported here support a model in which Nrp1, through its cytoplasmic domain and independently of its activity as a SEMA3A and VEGF-A165 coreceptor, stimulates GIPC1/Myo6-dependent endocytosis and traffic of active α5β1 integrin, thus promoting EC adhesion to FN and FN fibrillogenesis.

27In rescue experiments, where we reintroduced full-length and mutant murine Nrp1 constructs in human ECs in which endogenous hNrp1 was simultaneously knocked down by RNAi, we showed that EC adhesion to FN and polymerization of endogenous sFN into fibrils depend on the cytoplasmic domain of Nrp1, the C-terminal SEA motif representing the minimal sequence required to exert these functions. Importantly, as already shown for SEMA3A-elicited growth cone collapse in neurons, we found that the cytoplasmic domain of Nrp1 is instead dispensable for VEGF-A165 stimulation and SEMA3A inhibition of EC adhesion to FN. Moreover, by using two CHO cell clones differing in the expression of α5β1 integrin, we demonstrated that Nrp1 alone does not directly mediate adhesion to FN and that it requires α5β1 integrin. Therefore, we conclude that in ECs, independently of VEGF-A165 and SEMA3A, Nrp1 stimulates α5β1-mediated adhesion to FN and endogenous FN fibrillogenesis via its cytoplasmic SEA motif. This motif, similar to the C-terminal SDA sequence of the α5 integrin subunit, selectively and specifically binds the PDZ domain of the homomultimeric endocytic adaptor GIPC1.

28It is known that conformational activation of cell-surface integrins supports cell adhesion and spreading, whereas transition of integrins toward an inactive bent conformation causes cell de-adhesion and rounding up. However, we observed that lack of Nrp1 does not result in the reduction of either active or total α5β1 integrin either at the cell surface or intracellularly. Rather, by combining biochemical analysis with conventional and TIRF/FLIM confocal microscopy, we found that at the plasma membrane Nrp1 is tightly associated with adhesion sites, where it physically interacts with α5β1 integrin. The complex formed between active α5β1 integrin and Nrp1 is then rapidly internalized into Rab5-positive endosomes in an Nrp1-dependent fashion. Interestingly, the integrin is then returned to the plasma membrane from Nrp1-containing vesicles, and this recycling event appears to be targeted to adhesive structures. In addition, although the extracellular domain of Nrp1 is sufficient for its interaction with α5β1 integrin, the C-terminal GIPC1-binding SEA sequence of Nrp1 is necessary for stimulating EC adhesion to FN. Accordingly, knocking down either GIPC1 or its interacting motor Myo6 results in a significant impairment of active α5β1 integrin endocytosis and EC adhesion to FN.

29Taken together, our data indicate that, during EC adhesion and spreading on FN, Nrp1, through its extracellular domain, transiently interacts with active α5β1 integrin at adhesive sites and, via its cytoplasmic association with GIPC1, enhances the early endocytosis and the ensuing recycling of active α5β1 integrin to newly forming adhesion sites (Figure 11A). It is therefore likely that fast cycles of endocytosis from and recycling to ECM adhesions of active α5β1 integrin could allow real-time optimization of adhesion during EC spreading on FN. These conclusions are in line with the recent findings by Ivaska and colleagues that found how endocytosis of β1 integrins, in addition to their established role in directional migration, regulates cell adhesion and spreading as well. In particular, they reported that class V Rab GTPases (for review, see) Rab21 and Rab5 directly bind to several integrin α subunits, α5 included, by interacting with the conserved membrane proximal region GFFKR, which interestingly has been previously implicated in conformational integrin activation. It is thus conceivable that GIPC1 oligomers could favor α5β1 integrin endocytosis by bridging the α5 integrin subunit and the Rab5/Rab21 interactor APPL1, finally stabilizing the interaction between these small GTPases and α5β1 integrin. This could represent a main functional feature distinguishing α5β1 from other integrin heterodimers not interacting with GIPC1. Finally, the fact that by 2 min after activation α5-PA-GFP disappeared from preexisting adhesion sites into vesicles without a concomitant cell retraction suggests the existence of a steady endo-exocytic flow of (active) α5β1 integrins from and toward existing ECM adhesions as well (Figure 11B). This mechanism could allow adherent cells to be always ready to rapidly exchange integrins among cell–ECM contacts in response to extracellular stimuli. Such a scenario is also compatible with a previous study by Ezratty and colleagues and implies that disassembly of ECM adhesions could depend on an imbalance of endocytosis over recycling.

30Our observation that Myo6 siRNA severely impairs EC adhesion to FN and results in a significant reduction in the internalization of active α5β1 integrin suggests that Myo6 cooperates with GIPC1 (Figure 11A and 11B) and is compatible with the notion that Myo6 plays a role in the formation and transport of endocytic vesicles along F-actin microfilaments. The decrease in FN mRNA that we noticed in sihMyo6 ECs is likely due to the lack of the transcriptional activity displayed by Myo6 in the nucleus that could depend on a still not fully characterized actin–myosin-based mechanism of transcription. Therefore, Myo6 can support EC adhesion and motility by promoting both active α5β1 integrin traffic (Figure 11A and 11B) and FN1 gene transcription (Figure 11C). Additionally, these findings can have significant implications for the biology of α5β1-expressing human carcinomas, in which Myo6 can be overexpressed and promote metastatic invasion.

31In conclusion, we propose here that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on FN by increasing the Rab5/GIPC1/Myo6-dependent internalization of active α5β1 integrin. Nrp1 modulation of α5β1-mediated adhesion can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice. We anticipate that signaling pathways controlling Nrp1 expression in ECs could ultimately modulate the activity of α5β1 integrin. In particular, Nrp1 is a major target of the inhibitory Delta-like 4–Notch signaling pathway that negatively regulates the formation of endothelial tip cells. Higher expression of Nrp1 in tip ECs compared with that in stalk ECs of angiogenic sprouts could differentially modulate α5β1 integrin traffic, thus favoring tip cell adhesion and spreading on FN. Finally, both Nrp1 and α5β1 integrin are expressed in pericytes and vascular smooth muscle cells, which have been implicated in vascular remodeling by intussusceptive angiogenesis. Further work is needed to assess whether Nrp1 is regulating α5β1 integrin function not only in ECs but also in pericytes and vascular smooth muscle cells.

32Goat polyclonal anti-Nrp1 (C-19) and rabbit polyclonal anti-β-tubulin (H-235) were from Santa Cruz Biotechnology. Mouse monoclonal anti-human-Nrp1 (MAB 3870) was from R&D Systems. Mouse monoclonal anti-FN (MAB88904) and anti-αvβ3-integrin (MAB1976), goat polyclonal anti-α5β1-integrin (AB1950), rabbit polyclonal anti-α5-integrin (AB1928), rabbit polyclonal anti-α2-integrin (AB1936), and anti-α3-integrin (AB1920) were from Chemicon. Mouse monoclonal anti-human-vinculin (V9131) and rabbit polyclonal anti-Rab5 (R4654) were from Sigma-Aldrich. Rat monoclonal anti-HA (3F10) was from Roche. Rabbit polyclonal anti-GFP (A11122) and 4′,6-diamidino-2-phenylindole (DAPI) were from Molecular Probes. Goat polyclonal anti-GIPC1 (ab5951) and rabbit polyclonal anti-Myo6 (ab11096) were from Abcam. Streptavidin–horseradish peroxidase was from Amersham. Mouse monoclonal anti-active-α5-integrin, SNAKA51, was previously described.

33Human plasma FN was from Tebu-bio. Human plasma vitronectin, Engelbreth-Holm-Swarm murine sarcoma laminin, and calf skin collagen type I were from Sigma-Aldrich. Recombinant human VEGF-A165 was from Invitrogen. Recombinant human SEMA3A and mouse Sema3F were from R&D Systems. Sulfo-NHS-SS-Biotin was from Pierce.

34Hemagglutinin-tagged mNrp1 deletion constructs were generated by standard PCR protocols according to the Taq polymerase manufacturer's instructions (Fynnzymes) and using an HA-tagged version of full-length mNrp1 kindly donated by A. Püschel (Westfälische Wilhelms-Universität, Münster, Germany) as template. Cytoplasmic domains and the last three amino acids SEA were deleted using the following oligonucleotide primers: (i) 5′-cgccatggagagggggctgccgttg-3′ (Fw); (ii) 5′-ccaacaggcacagtacag-3′ (Re1) to amplify the mNrp1 deleted of the cytoplasmic domain; (iii) 5′-gtaattactctgtgggttc-3′ (Re2) to amplify the mNrp1 deleted of the three amino acids SEA. The corresponding PCR product was first thymidine–adenine (TA)-cloned into pCR2.1TOPO (Invitrogen) and subsequently subcloned in PINCO retrovirus or pAcGFP-N1 Vector (BD Bioscience) whose GFP coding sequence was previously substituted with the cDNA of mRFP, a kind gift of R. Tsien (University of California, San Diego, CA). α5-GFP and Rab5-GFP constructs were kindly provided, respectively, by A.F. Horwitz (University of Virginia, Charlottesville, VA) and M. Zerial (Max Plank Institute of Molecular Cell Biology and Genetics, Dresden, Germany).The α5-PA-GFP construct was previously described.

35The day before oligofection, ECs were seeded in six-well dishes at a concentration of 10 × 104 cells/well. Oligofection of siRNA duplexes was performed according to the manufacturer's protocols. Briefly, human ECs were transfected twice (at 0 and 24 h) with 200 pmol of siCONTROL nontargeting siRNA (as control), siGENOME SMART pools (in the case of hGIPC1 and hMyo6), or a mix of three (in the case of hNrp1) siRNA oligonucletides (Dharmacon). After 24 h (in the case of hNrp1) or 96 h (in the case of hGIPC1 or hMyo6) had passed since the second oligofection, ECs were lysed or tested in functional assays. In the case of hNrp1, the single oligonucleotide sequences were: (1) 5′-AAUCAGAGUUUCCAACAUA-3′; (2) 5′-GAAGGAAGGGCGUGUCUUG-3′; (3) 5′-GUGGAUGACAUUAGUAUUA-3′.

36Six-thousand ECs were resuspended in 0.1 ml of EBM-2 (Clonetics) with or without appropriate stimuli (50 ng/ml VEGF-A, 200 ng/ml SEMA3A, and 400 ng/ml SEMA3F) and plated on 96-well microtiter plates (Costar) that were previously coated with ECM proteins at different concentrations and then saturated with 3% bovine serum albumin. After 15 min of incubation at 37 °C, cells were fixed in 8% glutaraldehyde and then stained with 0.1% crystal violet in 20% methanol. Cells were photographed with a QICAM Fast 1394 digital color camera (QImaging) and counted by means of Image-ProPlus 6.2 software (Media Cybernetics). In adhesion assays with NIH 3T3 fibroblasts or CHO cells, Dulbecco's modified Eagle's medium was used.

37Endothelial cells were lysed in buffer containing 25 mM Tris-HCl, pH 7.6, 100 mM NaCl, 0.15% Tween-20, 5% glycerol, 0.5 mM ethylene glycol tetraacetic acid (EGTA), and protease inhibitors (50 mg ml−1 pepstatin; 50 mg ml−1 leupeptin; 10 mg ml−1 aprotinin; 2 mM phenylmethanesulfonylfluoride (PMSF); 2 mM MgCl2). Cells were lysed in buffer, incubated for 20 min on wet ice, and then centrifuged at 15,000g, 20 min, at 4 °C. The total protein amount was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce). Equivalent amounts (1,200 μg) of protein were immunoprecipitated for 1 h with the antibody of interest, and immune complexes were recovered on Protein G-Sepharose (GE Healthcare). Immunoprecipitates were washed four times with lysis buffer, twice with the same buffer without Tween-20, and then separated by SDS-PAGE. Proteins were then transferred to a Hybond-C extra nitrocellulose membrane (Amersham), probed with antibodies of interest, and detected by an enhanced chemiluminescence technique (PerkinElmer).

38Integrin traffic assays were performed as previously described by Roberts et al. with minor modifications. Cells were transferred to ice, washed twice in cold phosphate-buffered saline (PBS), and surface-labeled at 4 °C with 0.2 mg/ml sulfo-NHS-SS-biotin (Pierce) in PBS for 30 min. Labeled cells were washed in cold PBS and transferred to prewarmed EGM-2 at 37 °C. At the indicated times, the medium was aspirated, and dishes were rapidly transferred to ice and washed twice with ice-cold PBS. Biotin was removed from proteins remaining at the cell surface by incubation with a solution containing 20 mM sodium 2-mercaptoethanesulfonate (MesNa) in 50 mM Tris-HCl (pH 8.6), 100 mM NaCl for 1 h at 4 °C. MesNa was quenched by the addition of 20 mM iodoacetamide (IAA) for 10 min, and after other two further washes in PBS, the cells were lysed in 25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 1 mM Na3VO4, 0.5 mM EGTA, 1% Triton X-100, 5% glycerol, protease mix (Sigma), and 1 mM PMSF. Lysates were cleared by centrifugation at 12,000g for 20 min. Supernatants were corrected to equivalent protein concentrations by BCA assay, and integrins were isolated by immunoprecipitation and analyzed by SDS-PAGE.

39Cells were placed in RNAlater solution (Ambion), kept at 4 °C for 24 h, and frozen at −80 °C. After the cells were thawed on ice, total RNA was extracted following the manufacturer's recommended protocol (SV Total RNA Isolation System, Promega). The quality and integrity of the total RNA were quantified by means of the RNA 6000 Nano Assay kit in an Agilent 2100 bioanalyzer (Agilent Technologies). cDNAs were generated from 1 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).

40mRNA expression of FN and endogenous control genes, i.e., 18S rRNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and TATA binding protein (TBP), was measured in the samples by real-time RT-PCR using TaqMan Gene Expression Assays run on an ABI PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems). The following assays were used: Hs00365058_m1 (FN), Hs99999901_s1 (18S rRNA), Hs99999905_m1 (GAPDH), and Hs00427620_m1 (TBP). Three replicates were run for each gene for each sample in a 384-well format plate (cDNA concentration 20 ng/well) according the manufacturer's protocol. Between the three measured endogenous control genes, we chose TBP for normalization, identified by geNorm. The experimental threshold (Ct) was calculated using the algorithm provided by the SDS 1.9.1 software (Applied Biosystems). Ct values were converted into relative quantities using the method described here. The amplification efficiency of each gene was calculated using a dilution curve and the slope calculation method.

41Cells were plated on glass coverslips coated with 1 μg/ml FN (TebuBio) and allowed to adhere for 3 h. In addition, ECs cotransfected with mNrp1-mRFP and Rab5-GFP were then washed in PBS, incubated with 10 μg/ml SNAKA51 Ab in EBM-2 for 30 min at 4 °C, washed 3 times in PBS, transferred to prewarmed EGM-2, and allowed to recover at 37 °C for 2 min to induce endocytosis. Cells were washed in PBS, fixed in 4% paraformaldehyde, permeabilized in 0.01% saponin for 10 min on ice, and incubated or not with the Alexa-Fluor-405-conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. Cells were analyzed by using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems). Immunofluorescence analysis was performed as previously described.

42Small interfering RNA silencing was performed, and after the second oligofection, cells were seeded onto glass coverslips in six-well dishes at a concentration of 20 × 104 cells/well and left to adhere for 3 h in EGM-2 medium (Clonetics) containing FN-depleted serum. Cells were then washed with PBS and fixed with 3.7% paraformaldehyde for 20 min at room temperature. Next, cells were permeabilized in PBS containing 0.1% Triton X-100 on wet ice for 2 min and incubated with anti-FN Ab for 1 h at room temperature. After three washes, cells were incubated with anti-mouse Alexa Fluor 555 for 45 min at room temperature and subsequently with DAPI. Cells were finally examined using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems).

43Fluorescence resonance energy transfer was detected using a Lambert Instruments fluorescence attachment (LIFA) on a Nikon Eclipse TE 2000-U microscope, with the same changes to the condenser as described above and a filter block consisting of a Z473/10 excitation filter, a Z 488 RDC dichroic mirror, and a HQ 525/50M emission filter. The light source was a modulated 473-nm laser diode, which allows, in combination with the modulated intensifier from the LIFA system, measurement of fluorescence lifetimes using frequency domain. The laser was brought into TIRF mode before acquiring the images for the lifetime analysis.

44Donor (D) lifetime, τ, was analyzed either in the presence or in the absence of the acceptor (A), in adhesion sites, characterized by high donor concentrations, using the FLIM software (version 1.2.1.1.130; Lambert Instruments, The Netherlands). Fluorescence resonance energy transfer efficiency (E) was calculated as E = 1 – (τDA/τD). Lifetime τ was evaluated in four different areas (12 × 12 pixels) of seven α5-GFP and seven α5-GFP/mNrp1-Cherry transfected NIH 3T3 cells. For statistical evaluation, results were analyzed with Student's t test.

45Total internal reflection fluorescence experiments have been performed on a Nikon Eclipse TE 2000-U microscope equipped with 60× and 100× 1.45 NA Nikon TIRF oil immersion objectives. The Nikon Epi-fluorescence condenser was replaced with a custom condenser in which laser light was introduced into the illumination pathway directly from the optical fiber output oriented parallel to the optical axis of the microscope. The light source for evanescent wave illumination was either a 473-nm diode, a 405-nm diode, or a 561-nm laser (Omicron), with each laser line coupled into the condenser separately to allow individual TIRF angle adjustments. Each laser was controlled separately by a DAC 2000 card or a uniblitz shutter operated by MetaMorph (Molecular Devices). A filter block consisting of an E480SPX excitation filter, a FF 495 dichroic mirror, and an ET 525/50M emission filter was used for activation of α5-PA-GFP with the 405-nm laser. After activation the filter was manually changed to a green/red dual filter block (ET-GFP/mCherry from AHF Analysentechnik, Germany) to allow simultaneous time-lapse acquisition of activated α5-PA-GFP and mNrp1-Cherry using 473- and 561-nm excitation. A Multi-Spec dual emission splitter (Optical Insights, NM) with a 595-nm dichroic and two bandpass filters (510–565 nm for green and 605–655 nm for red) was used to separate both emissions. All cell imaging was performed with a Cascade 512F EMCCD camera (Photometrics UK).

46Localized activation of α5-PA-GFP in mNrp1-Cherry-positive vesicles was done on a FV 1000 Olympus confocal microscope, using two-channel imaging and a separate SIM scanner for 405-nm activation.