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. 2009 Mar 3;17(5):837–843. doi: 10.1038/mt.2009.29

Clustering and Internalization of Integrin αvβ3 With a Tetrameric RGD-synthetic Peptide

Sancey Lucie 1,2, Garanger Elisabeth 1,2,3, Foillard Stéphanie 2,3, Schoehn Guy 4,5, Hurbin Amandine 1,2, Albiges-Rizo Corinne 2,6,7, Boturyn Didier 2,3, Souchier Catherine 2,8, Grichine Alexeï 2,8, Dumy Pascal 2,3, Coll Jean-Luc 1,2
PMCID: PMC2760123  PMID: 19259068

Abstract

Integrin αvβ3 is overexpressed on neoendothelial cells and frequently on tumor cells. We have developed a peptide-like scaffold (regioselectively addressable functionalized template, RAFT), which holds four cyclo(-RGDfK-) (cRGD) motifs and proved that this molecule (called regioselectively addressable functionalized template-arginine-glycine-aspartic acid, RAFT-RGD) targets integrin αvβ3 in vitro and in vivo. Using fluorescence correlation spectroscopy (FCS), we measured the constant of affinity (KD) of the RAFT-RGD for purified integrins. KD values rose from 3.87 nmol/l for RAFT-RGD to 41.70 nmol/l for cyclo(-RGDfK-). In addition, RAFT-RGD inhibited αvβ3 lateral mobility in the cell membrane, probably due to the formation of integrin clusters as demonstrated by fluorescence recovery after photobleaching (FRAP). This was confirmed by electronic microscopy data, which established the formation of molecular complexes containing two integrins in the presence of RAFT-RGD but not cRGD or regioselectively addressable functionalized template-arginine-alanine- aspartic acid (RAFT-RAD). Using an enzyme-linked immunosorbent assay (ELISA), we proved that 1 µmol/l RAFT-RGD increased by 79% αvβ3 internalization via clathrin-coated vesicles. Conversely, cRGD was internalized without modifying αvβ3 internalization. Although RGD has been known for >20 years, this is the first study to formerly establish the relationships among multimeric presentation, increased affinity, and subsequent integrin-mediated cointernalization. These results strongly support the rationale for using multimeric RGD-peptides as targeting vectors for imaging, diagnosis, or therapy of cancers.

Introduction

Targeting tumor angiogenesis for specific drug transfer into tumor masses and metastasis has been identified as a promising approach for three main reasons: (i) angiogenesis is a common and genetically stable characteristic of most solid tumors, (ii) it is readily accessible from the blood stream, and (iii) it can be targeted by specific arginine-glycine-aspartic acid (RGD)-containing peptides binding integrin αvβ3. This integrin is indeed poorly expressed on quiescent vessels and is selectively overexpressed on activated endothelial cells of growing vessels. In addition, integrin αvβ3 is also frequently overexpressed on tumor cells, as observed in lung cancers,1 melanomas,2 brain tumors,3 or breast cancers.4

Integrins are membrane-spanning heterodimers of α and β subunits, both of which comprise a short cytoplasmic tail, a single transmembrane helix, and a large extracellular domain.5 Most integrins are expressed in a default low-affinity ligand-binding state but their conformation and affinity can vary in response to cellular and microenvironment stimulations.6,7 This will also affect their lateral assembly and clustering on the surface of the cell.8 Several groups have developed multimeric RGD-presenting molecules, with the aim of not only to increase integrin affinity and clustering but also to induce an active integrin-mediated internalization.9,10,11

We have developed a regioselectively addressable functionalized template (RAFT) cyclo-decapeptide scaffold, able to present four cyclic RGD pentapeptide motifs. We have shown using nuclear or optical imaging methods, that regioselectively addressable functionalized template-arginine-glycine-aspartic acid (RAFT-RGD) allows an improved and αvβ3-specific targeting and in vivo imaging of tumors as compared to the monomeric cyclic RGD (cRGD).12,13,14,15

Surprisingly, although the interaction between RGD-ligands and integrins has been known for a long time16,17 and RGD-containing molecules have been widely used to deliver various kinds of cargos including nanoparticles (liposomes or polymers), cytotoxic peptides, low molecular weight drugs, and contrast-enhancing agents (fluorochromes, radiotracers),11,18,19,20 very little is known about the internalization mechanism of RGD-peptides binding to integrin αvβ3.

Two studies have described how an antibody directed against integrin αvβ3 (mAb 17E6) and monomeric or multimeric RGD-peptides is internalized. Both concluded that the internalization of monomeric RGD-ligands is independent of its αvβ3 receptor and occurs via a fluid-phase endocytic pathway. In contrast, multimeric RGD molecules are cointernalized with their receptor,21,22 evidence in favor of integrin aggregation and clustering.

The integrin endo/exocytic cycle23,24,25 suggests that there are, at least, three types of pathways associated with integrin internalization: (i) clathrin-mediated endocytosis was described for αvβ5 integrins;26 (ii) caveolae-mediated endocytosis for α2 integrins,27 and (iii) clathrin–caveolae-independent endocytosis. To our knowledge, endocytosis of αvβ3 integrins was described to occur through clathrin-dependent endocytosis28 or uncoated vesicles.29 Clathrin-dependent endocytosis has also been described for viruses such as adenoviruses that enter the cells after binding to the αvβ3 integrin secondary receptor.30

Here, we studied the biological properties of the tetrameric RAFT-RGD peptide (coupled or not to fluorescent probes) as compared to those of its monomeric counterpart cRGD so as to better understand and improve the potential of RGD-based vectors to specifically deliver therapeutic drugs directly inside the target cells.

Results

Affinities of RAFT-RGD versus cRGD for the purified, soluble integrin αvβ3

Fluorescence correlation spectroscopy (FCS) is commonly used to characterize the dynamics of fluorescent molecules in solution. This technique allows users to measure fluorescence intensity fluctuations due to diffusion phenomenon, chemical reactions, aggregation, etc. We first established the diffusion properties of each fluorescently labeled RGD-containing molecules in solution and then measured the variation of this parameter in the presence of a large excess of purified integrins. This provided quantitative information allowing the determination of a constant of association (KD).

FCS analysis indicated that the tetrameric RAFT-RGD-Cy5 had a tenfold higher affinity for its soluble receptor integrin αvβ3 in Hank's buffered salt solution (containing Ca2+/Mg2+) than the monomeric cRGD-Cy5 (Figure 1b). Its KD, obtained by curve fitting with a two-component model, was 3.87 nmol/l while the KD of cRGD-Cy5 reached 41.70 nmol/l. The nonspecific RAFT-RAD-Cy5 did not interact with integrin αvβ3: the data fitted neither a two- nor even a three-component model but only fitted a one-component model, corresponding to free RAFT-RAD-Cy5. The KD of RAFT-RGD-Cy5 was also determined for a nonspecific receptor integrin α3β1: in this case, the measured KD was 1,147 nmol/l, i.e., 300-fold higher than the one obtained with αvβ3.

Figure 1.

Figure 1

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RGD-peptides' affinities. (a) Chemical structures of RGD-peptides. The monovalent cyclo(-RGDfK-) (cRGD) was compared with the tetrameric RAFT(c(-RGDfK-)4) (RAFT-RGD). cRGD was modified on the lysine side chain to obtain the fluorochrome-conjugated cRGD-FITC or cRGD-Cy5. For RAFT-RGD, fluorochromes were conjugated to the lower face of the RAFT scaffold (central alanine residues replaced by lysine). RAFT(c(-RβADfK-)4) (RAFT-RAD) was used as negative control. (b) FCS analysis of the interaction of Cy5-labeled peptides with soluble integrins at 633 nm. KD was determined at the equilibrium. The diffusion time τD and the diffusion coefficients D of the peptides alone or in a complex with the integrin are indicated. Data were best-fitted by a two-components model and are represented as mean ± SD. Representative plots are available in the Supplementary Figure S1.

RAFT-RGD slows down integrin αvβ3 mobility in the cell membrane

We measured the mobility of integrin αvβ3 in the membrane of adherent human embryonic kidney 293(β3) (HEK293(β3)) cells in the presence of the different peptides by fluorescence recovery after photobleaching (FRAP) analysis using a confocal microscope. These cells expressing natural amount of the human αv chain were stably transfected with a plasmid encoding for the human β3 chain. We focused the laser beam on the apical membrane for two reasons: (i) integrins from this region are mobile because they are not engaged in cell–matrix adhesions and (ii) this area is less affected by cell shrinkage usually observed in the presence of RGD-peptides. Adherent cells were coincubated for 8 minutes with the different peptides and the R-Phycoerythrin (RPE)-labeled LM609 antibody. After the RPE photobleaching in a defined area (region of interest, ROI), the time for fluorescence recovery due to the lateral moving of RPE-labeled integrins on the membrane was measured (Figure 2). Importantly, we initially verified that LM609's binding was not affected by the presence of the peptides (data not shown). Also, no significant cellular movement or changes in membrane curvature occurred during fluorescence sampling. Results presented in Figure 2 indicate that the presence of RAFT-RGD-fluorescein isothiocyanate (FITC) peptide dramatically slowed down the recovery of the integrin signal into the bleached area as compared to untreated cells. In contrast, no decrease in the time of recovery was observed either by using the negative control peptide RAFT-RAD-FITC or with the monovalent cRGD-FITC. The apparent diffusion time, calculated from the fluorescence recovery curves obtained on 20 individual cells (three separated experiments), increased from 46 ± 14 seconds (RAFT-RAD-FITC, cRGD-FITC, and phosphate buffered saline) to 144 ± 22 seconds (RAFT-RGD-FITC). No concomitant change in peptide distribution was induced during FRAP experiments as monitored in the FITC detection channel. The fluorescence recovery being directly correlated to the mobility of the receptor, these results suggested that the presence of the tetrameric RAFT-RGD-FITC slowed down αvβ3 integrin diffusion within the cell membrane. Several hypothesis may explain this result among which we favor: the multimeric RAFT-RGD crosslinks several integrins or RAFT-RGD modifies the activation state of the integrin, which in turn would be more strongly linked to the cytoskeleton.

Figure 2.

Figure 2

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RAFT-RGD-FITC reduces αvβ3 integrin lateral mobility. R-Phycoerythrin-conjugated LM609 monoclonal antibody was used for direct observation of αvβ3 integrin diffusion on the apical membrane of the cell. Adherent HEK293(β3) cells were incubated with 0.5 µmol/l FITC-labeled RAFT-RGD or RAFT-RAD or 2 µmol/l cRGD-FITC or in absence of peptides. The antibody LM609-RPE was also present during the 8 minutes of incubation with the peptides in order to follow integrin lateral diffusion. After washing, the cells were observed on an inverted confocal microscope. Recovery of the integrin signal into the bleached area is significantly slowed down in the condition where cells were incubated in the presence of the multimeric RGD-presenting ligand, RAFT-RGD-FITC, as compared to nontreated cells, or to RAFT-RAD-FITC or cRGD-FITC treated cells.

RAFT-RGD can bind two αvβ3 integrins simultaneously

In order to comfort the data obtained by FRAP, we aimed at visualizing the possible formation of integrin clusters induced by RAFT-RGD. We used negative staining electron microscopy to observe αvβ3 integrin and αvβ3/RGD-peptides mixed in 1 mmol/l Mg2+/Ca2+ as illustrated in Figure 3. Peptides were used in excess as compared to the integrin concentration. These conditions are not supposed to maximize the number of dimers.31,32 Integrins alone or mixed with cRGD or RAFT-RAD displayed compact particles representing single heterodimers of αvβ3 ± RGD as expected. Indeed, the monomeric cRGD does not have the possibility to interact with several integrins at the same time, while the RAFT-RAD is not able to recognize them at all. Conversely, αvβ3/RAFT-RGD micrographs were frequently showing larger particles corresponding to complexes of two αvβ3 integrins probably linked by the multimeric RAFT-RGD. Visually, we estimated that about 10% of αvβ3/RAFT-RGD were forming integrin dimers. But, this percentage was most probably underestimated because we counted only the aggregates laying on the grid in a proper angle of examination and providing particles with this typical dimer-shape. For example, clusters viewed down the long axis would appear more compact33 and were not included (the staining agent outlines only those parts of the objects that are in contact with the carbon film). Nevertheless, electron microscopy is a qualitative technique and not a quantitative technique: dimers of integrins may bind with less affinity to the carbon than monomeric integrins, thus leading to underestimate the number of dimers.

Figure 3.

Figure 3

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RGD-peptides/integrin αvβ3 complexes. Representative examples of negatively stained electron micrographs of the soluble αvβ3 integrin alone or mixed with RGD-peptides. The αvβ3 integrins remain in monomeric state except when using RAFT-RGD; in this condition, integrins can be found as dimers on the grid. As expected, RGD-peptides (<6 kd) were not distinguishable. Upper panels: original images. Lower panels: photoshop enhanced visualization of the complexes.

RAFT-RGD-mediated integrin αvβ3 internalization

RAFT-RGD-Cy5 and cRGD-Cy5 internalizations were observed by confocal microscopy on live HEK293(β3) cells (Figure 4). RAFT-RGD-Cy5 was rapidly internalized in small vesicles after 10 minutes (Figure 4a) but was also found in the cytoplasm and at cell–cell contacts. Monomeric cRGD-Cy5 internalization is less extensive than that of RAFT-RGD-Cy5 and the laser intensity had to be increased three times in order to obtain comparable signal intensities (Figure 4e).

Figure 4.

Figure 4

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Confocal imaging on HEK293(β3) living cells. Cells were starved for 30 minutes and incubated with 1 µmol/l RAFT-RGD-Cy5 (a–d) or 1 µmol/l cRGD-Cy5 (e–h) for 10 minutes at room temperature in Dulbecco's modified Eagle's medium medium alone (a,e) or containing amantadine 1 mmol/l (b,f), nystatin 1 µmol/l (c,g) or amiloride (d,h) 1 mmol/l. Cells were then rinsed and observed at 633 nm. Peptide internalization was evaluated according to the Cy5 intensity in the cells and is indicated for each photo. Bar = 10 µm.

We then developed a special enzyme-linked immunosorbent assay (ELISA) to demonstrate that RGD-peptides were inducing integrin αvβ3 internalization. Briefly, integrins exposed on the surface of HEK293(β3) cells were biotinylated and the cells were incubated in the presence of 0–1 µmol/l RAFT-RGD or 0–4 µmol/l cRGD for 10 minutes in order to keep the number of RGD motifs constant. The cells were then lysed, fractionated, and the concentration of biotinylated-αvβ3-integrins present into each fraction measured using ELISA. The absence of peptide (control condition) established the normal endocytosis of integrin αvβ3; we found that 12 ± 1% of the labeled integrins are internalized “naturally” in 10 minutes (Table 1). In the presence of RAFT-RGD, internalization increased in a dose-dependent manner and reached 21 ± 2% at 1 µmol/l, corresponding to an increase of 79% versus control. In contrast, increasing doses of cRGD (from 0.1 to 4 µmol/l) did not affect integrin internalization at all, which remained similar to that of the control (i.e., 12 ± 1% at 1 µmol/l).

Table 1.

Integrin αvβ3 internalization assay

graphic file with name mt200929t1.jpg

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Altogether this indicated that RAFT-RGD internalization was correlated with integrin αvβ3 endocytosis, although the monomeric cRGD did not affect αvβ3 natural endocytosis.

RAFT-RGD internalization occurs via clathrin-mediated endocytosis

RAFT-RGD-Cy5 and cRGD-Cy5 internalization pathways were analyzed using confocal microscopy in the presence of specific inhibitors (Figure 4). In the presence of the clathrin-inhibitor amantadine at 1 mmol/l (Figure 4b), RAFT-RGD-Cy5 internalization was extensively inhibited. The fluorescence was found at the cell surface mainly and especially at the cell–cell contacts. In contrast, amantadine did not affect cRGD-Cy5 internalization (compare Figure 4e,f). One µmol/l of nystatin, an inhibitor of caveolae-dependent internalization had no effect on either peptide (Figure 4c,g). In the presence of 1 mmol/l amiloride, internalization of the peptides remained unchanged (Figure 4d,h) although, we proved by using 70 kd-dextran-FITC that macropinocytose was correctly inhibited in these cells (data not shown).

These results were confirmed by the ELISA measurements of the integrin αvβ3 amount internalized after binding to RAFT-RGD in the presence of amantadine, nystatin, or amiloride (Table 1). In the presence of amantadine, 1 µmol/l of RAFT-RGD was not able any more to induce integrin internalization and the % of internalized integrins was exactly similar to the control values (12 ± 2%). In contrast, nystatin or amiloride did not prevent RAFT-RGD-induced integrin αvβ3 internalization. These data suggested that RAFT-RGD was internalized with integrin αvβ3 via the clathrin-dependent pathway. Furthermore, peptides' internalization was quantified from confocal microscopy analysis. The related peptide internalization indexes are reported in Figure 4 (mean of Cy5-intensity/pixels). Those indexes confirmed that RAFT-RGD internalization occurred in a clathrin-dependent pathway, but also that RAFT-RGD gets into the cells more efficiently than cRGD.

Discussion

Drug vectorization mediated by specific tumor-targeting molecules could allow specific delivery of cytotoxic agents to tumors therefore limiting their systemic toxicity. Based on this concept, RGD-containing peptides have been largely used for the targeting of αvβ3-integrin expressing tumors and/or of their microvasculature. Our group contributed to the development of a synthetic multimeric RGD-based vector, called RAFT-RGD. This peptide proved to be particularly efficient for the delivery of drugs,34 imaging agents, or both.12,13,14,15,35 However, although RGD–integrin interaction has been discovered a long time ago,16,17 the mechanism of internalization of monomeric or multimeric RGD-peptides is a poorly documented process. In this study, we focused our attention on the study of the mechanism by which the well-known cRGD, similar to the original cyclic peptide developed by Kessler et al.36,37 and its RAFT-supported tetrameric version RAFT-RGD are internalized, with a particular emphasis on the internalization pathways involved after recognition and binding to the αvβ3 receptor.

Multimeric RGD-peptides are expected to present an increased affinity for the αvβ3 integrin as compared to their monomeric counterpart. This has been demonstrated when comparing cyclic versus linear RGD-based peptides.38,39 We confirmed this characteristic for the RAFT-RGD using an FCS assay. RAFT-RGD-Cy5 bound specifically to integrin αvβ3 with a tenfold higher affinity than cRGD (Figure 1b). Surprisingly, although integrins are known to present at least two affinity states, we measured only one KD value for both RAFT-RGD-Cy5 and cRGD-Cy5. In addition, the KD value measured for cRGD was ~25 times higher than previously reported values.39,40 This suggested that, in our FCS assay, integrins were exclusively in their activated form as a result of the combined presence of octyl-β-D-glucopyranoside (unpublished observations), Mg2+ ions41 and of cRGD-peptides8,42 in the media, each of these factors being known to switch integrins in their high affinity state. In addition, it must be noticed that previous measurements of the KD described in the literature were based on solid-phase receptor binding assays. In our case, integrins were in solution and this certainly modified their constant of affinity.

Using FRAP, we also demonstrated that the multimeric RAFT-RGD decreased the lateral mobility of αvβ3 receptors on the surface of HEK293(β3) cells. This suggested that the presence of four cRGD motifs onto the RAFT scaffold allowed the clustering of integrin αvβ3. This result is important because it indicated that at least two cRGD motifs presented by a single RAFT molecule can bind two integrins. This was an open challenge for the RAFT scaffold, which is no more than 10 Å large. Indeed, the three-dimensional structure of purified αvβ3 integrin showed that the diameter of this integrin is close to 100 Å,40 but that the RGD binding site is on the periphery of the molecule. The two RGD motifs presented by a single RAFT could thus bridge two integrins positioned back to back. This was confirmed by EM results, which indicated that the tetrameric, but not the monomeric cRGD, could form clusters of two integrins.

Our estimation using EM indicates that around 10% of integrins can be crosslinked when large excess of RGD-peptides are used (their concentrations were 2–3 order of magnitude larger than their respective KD). This is not in agreement with theoretical models described by Dembo and colleagues,31,32 suggesting that crosslinking of these integrins by RAFT-RGD could also happen by other mechanisms than specific binding of the two RGD's. However, it is difficult to explain in this case why this nonspecific crosslinking would occur in this in vitro assay with RAFT-RGD and not with RAFT-RAD or cRGD. Thus, the theoretical model may not apply to these purified heterodimeric molecules extracted from their natural cellular environment, which, in addition, are not bivalent like the one used to describe the model. This may be different when live cells presenting finely tunable integrins are used. In this case, the activation of the integrin may differ in the presence of tetrameric versus monomeric RGD's. RAFT-RGD could activate the integrins and eventually trigger complex intracellular cascades that would augment the integrin–actin interaction and limit the lateral motility of the integrins on the cell surface. Thus, formation of clusters of integrins may not be the sole mechanism explaining the results we obtained using FRAP.

Anyhow, an active internalization of the tetrameric RAFT-RGD-Cy5 via, and concomitantly with integrin αvβ3 is observed. Indeed, the natural endocytosis of this integrin almost doubled in <10 minutes in the presence of RAFT-RGD and its internalization was mainly involving clathrin-mediated endocytosis. Accordingly, this process was abolished in the presence of amantadine, a specific inhibitor of the clathrin-mediated endocytosis. Macropinocytosis and caveolae-mediated endocytosis may not be implicated because their inhibitors like amiloride or nystatin had no effect on RAFT-RGD internalization. Interestingly, the monomeric cRGD-peptide interacted in a completely different manner. Its internalization did not rely on clathrin- or caveolae-mediated endocytosis and was most probably independent of αvβ3 because the internalization of the integrin was not affected. These results are in agreement with a previous report and indicated that cRGD can probably cross cell membranes via a fluid-phase pathway.22 The corresponding efficiency of internalization is, however, much less efficient than that of the RAFT-RGD, which explains the lower intensity of staining of the inside of cells labeled with cRGD-Cy5 and as demonstrated by the indexes in Figure 4.

Viruses such as foot-and-mouth disease virus43 or adenovirus present several RGD motifs allowing their interaction with the αvβ3 integrin. This interaction is a prerequisite to their internalization,30,44 which also occurs via clathrin-coated vesicles.45 RAFT-RGD may thus mimic some properties of these viruses.

In summary, our study establishes that the tetrameric RAFT-RGD binds 10 times more strongly to its αvβ3 receptor than cRGD, but also that it is actively and efficiently internalized with integrin αvβ3 via clathrin-coated pits as previously described for the αvβ3 integrin.46 This contrasts with the trafficking route followed by the β1 integrin, which was shown to use preferentially a caveolae-dependent pathway.47

From this study, multimeric presentation of cRGD motifs appears to be a prerequisite for the development of efficient integrin targeting and cell internalizing vectors for drug delivery to tumors.

Materials and Methods

Material. Integrin αvβ3 was purchased from Chemicon International, Temecula, CA (CC1021; St Quentin en Yvelines, France). Monoclonal antibody antihuman integrin αvβ3 LM609 conjugated to RPE (RPE-LM609) and antihuman CD61 were purchased from Chemicon and Beckman Coulter (IM0540; Villepinte, France), respectively. Cycloheximide was from Sigma-Aldrich (Lyon, France). NHS-SS-biotin was from Pierce (21441; Brebières, France).

RGD-peptides synthesis and fluorescent labeling. Compounds were synthetized according to previously reported procedures35 and chemical structures are presented in Figure 1a. Briefly, RAFT is a cyclic decapeptide (c(-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-Lys(Boc)-Lys(Alloc)-Lys(Boc)-Pro-Gly-)) having two orthogonally addressable domains pointing on either side of the cyclopeptide backbone. On the upper face, four copies of the c(-RGDfK-) peptide were grafted via an oxime bond (R1–O–N=C–R2) for recognition of the integrin αvβ3. On the other side of the RAFT, either Cy5 mono NHS (N-hydroxysuccinimide) ester (Amersham Biosciences, Uppsala, Sweden) or FITC (Sigma-Aldrich, St Quentin Fallavier, France) was added on the lysine chain (c(-KKKPGKAKPG-)).15 As a negative control probe, Cy5-labeled RAFT(c(-RβADfK-))4 (RAFT-RAD) was also synthesized in a similar way.

FCS analysis. FCS study was performed on the ConfoCor 2 system (Carl Zeiss, Jena, Germany) using a 40× water immersion C-Apochromat objective lens (numerical aperture (N.A.) = 1.2). The measurements were carried out at room temperature in 8-well Lab-Tek I chambered coverglass (Nalge Nunc International, Illkirch, France). The 633 nm He–Ne laser beam was focused into 50 µl solutions at 150 µm over the cover glass. The fluorescence emission was collected through a pinhole and a 650 nm-long pass filter. Photon counts were detected by an Avalanche PhotoDiode at 20 MHz for 30 seconds. For each sample, FCS measurements were repeated 15 times. The data evaluation was performed using the Zeiss FCS Fit software (Zeiss, Jena, Germany). Most of the intensity autocorrelation curves were fitted using a free diffusion model with two components: the peptide coupled to the fluorochrome alone and the fluorescent peptide–integrin complex. Preliminary studies enabled us to fix the diffusion time value of the first component and structural parameter. Moreover, a calibration step with 4 nmol/l Cy5 made it possible to evaluate the size of the confocal volume (≈1 fl). Interaction assays were performed at RT in HBSS containing Mg2+ and Ca2+. One to 40 nmol/l of soluble integrin αvβ3 (CC1021, Chemicon) were mixed with 0.6 nmol/l of RAFT-RGD-Cy5 and RAFT-RAD-Cy5 or 2.4 nmol/l of cRGD-Cy5. FCS measurements were performed 2 minutes after mixing. Theoretical calculation was made using Origin software (Origin Lab, Northampton, MA). The goodness-of-fit (χ2) was the mean end point for the quality of the fit (in our condition, 5 × 10−4 > χ2 > 1 × 10−6 for a good fit). Furthermore, the residual curves had no wavy shape (see example in Supplementary Figure S1).

Cell lines and culture conditions. HEK293(β3), stable transfectants of human β3 from the human embryonic kidney cell line (kindly provided by J.-F. Gourvest, Aventis, France), were cultured as described in Jin et al.13 The cell line was cultured at 37 °C in a humidified 95% air/5% CO2 atmosphere.

Confocal laser scanning microscopy and FRAP experiments. HEK293(β3) cells were grown for 24 hours on 18 mm round cover glasses placed in the wells of a 12-well plate (seeding density of 7 × 104 cells per well). Immediately before running the experiment, cells were incubated for 8 minutes at RT (22 °C) in a mixture of RPE-conjugated LM609 monoclonal antibody (RPE-LM609, Chemicon) and 0.5 µmol/l FITC-labeled RGD-peptides. The incubation with monovalent cRGD was performed at either 0.5 µmol/l or 2 µmol/l. The antibody and peptide solutions were extemporaneously prepared in Hank's buffered salt solution buffer enriched with 1 mmol/l MgCl2. For microscopic observations, coverslips were rinsed once in Hank's buffered salt solution buffer and disposed on a custom-made incubation chamber containing 200 µl of the FITC-labeled RGD-peptide solutions (0.5 µmol/l or 2 µmol/l with cRGD). The confocal imaging and FRAP measurements were carried out on an inverted confocal microscope (LSM510; Carl Zeiss) using a 40× water immersion objective of 1.2 N.A. A pinhole adjustment resulted in a 2.5 µm optical slice used for the visualization of a 25 µm circular region of the cell apex membrane at scan zoom 4. For FRAP experiments, a 3 µm circular ROI was uniformly bleached for 2 seconds with 100% intensity of the 543 nm line (fluorescence bleaching ratio >90%). The fluorescence recovery was then sampled on the whole region for 170 seconds every 5 seconds with 0.1% laser intensity set with acousto-optical tunable filters. Thanks to the extremely small excitation power and short acquisition times, no photobleaching was induced during sampling as observed on control cells or on the membrane out of the bleached ROI. Neither lateral nor axial displacement of ROI was observed during FRAP measurements and no recovery of fluorescence was observed on the entirely bleached control cells.

Data analysis was performed in assumption that the recovery of fluorescence in the ROI was solely due to the two-dimensional cytoplasmic diffusion of fluorescent species. The diffusion time τd was determined by fitting the normalized fluorescence recovery curves F(t) to the recovery kinetics equation:48,49

graphic file with name mt200929e1.jpg

where t is time, F0 and F∞ are initial and final mean fluorescence intensities after photobleaching, respectively, I0 and I1 are modified Bessel functions. The diffusion time values obtained for each peptide conditions are the mean of 20 individual cells.

Electron microscopy. Soluble human integrin αvβ3 (Chemicon, #CC1021) was diluted to 0.095 mg/ml (≈3.65 fmol) in PBS containing MgCl2 and CaCl2 at 1 mmol/l and mixed with RAFT-RGD 0.45 mg/ml (≈1 nmol), cRGD 0.1 mg/ml (≈1 nmol) or RAFT-RAD 0.45 mg/ml (≈1 nmol) for 2 minutes before addition on top of a carbon-coated electron microscope grid. Thirty seconds later, the excess of liquid was removed by blotting with a filter paper. Four milliliter of a 2% uranyl acetate solution were placed on the grid and incubated for 30 seconds to 1 minute at room temperature. The staining solution was subsequently removed by filter paper adsorption, and the grid was dried on a paper filter for 2 minutes and then examined using an electron microscope. Micrographs were taken under low-dose conditions with a Jeol 1200-EX II microscope (Jeol, Croissy- sur-seine, France) at 100 kV or an FEI CM12 microscope (Philips, Eindhoven, the Netherlands) at 120 kV and a, respectively, calibrated magnification of ×40,000 and ×45,000. Selected negatives films were digitalized on a Zeiss scanner (Photoscan TD) with a pixel size of 14 mm, corresponding to 3.5 Å or 3.1 Å at the sample scale.

Confocal microscopy of peptide internalization. HEK293(β3) cells were grown as described in 4-wells Lab-Tek I chambered coverglass. Cells were starved 30 minutes and incubated with Dulbecco's modified Eagle's medium w/o red phenol alone or containing amantadine 1 mmol/l, nystatin 1 µmol/l, or amiloride 1 mmol/l for another 30 minutes at 37 °C, 5% CO2. Then, 1 µmol/l RAFT-RGD-Cy5 or 1 µmol/l cRGD-Cy5 were added to the culture medium, together with 5 µmol/l of Hoechst, for 10 minutes. Confocal microscopy was performed on the Axiovert 200 LSM510 LNO Meta microscope (Carl Zeiss) using a 40× oil immersion objective of 1.2 N.A., after addition of fresh medium. The 633 nm laser intensity was set up on request at 10 or 30% of its maximum intensity depending on the peptide. The following inhibitors were used in order to block caveolae, clathrin-coated pits or macropinocytosis: nystatin 1 µmol/l, amantadine 1 mmol/l, or amiloride 1 mmol/l (Sigma-Aldrich).

Peptides internalization was quantified from confocal microscopy analysis. The mean Cy5 intensity was related to the cell area (in pixel); the related index is reported on the pictures.

Integrin internalization assay. Surface biotin labeling and internalization. HEK293(β3) cells were cultured at ~85% confluence in 90 mm dishes and starved at 37 °C for 30 minutes in Dulbecco's modified Eagle's medium containing 15 µmol/l of cycloheximide (Sigma-Aldrich, St Quentin Fallavier, France). Membrane labeling was adapted from Roberts et al.50 Depending of the condition, cells were kept at 4 °C or placed 10 minutes at 37 °C in Dulbecco's modified Eagle's medium alone (control) or containing RAFT-RGD or cRGD from 0.1 to 4 µmol/l, in order to allow receptor internalization.

In order to measure endocytosis inhibition, amantadine 1 mmol/l, nystatin 1 µmol/l, or amiloride 1mmol/l were added to the medium 30 minutes before biotin labeling. Those inhibitors were kept in the medium during biotin labeling and peptides internalization.

ELISA. Integrin internalization was quantified using ELISA in 96-wells plate, through gentle agitation. The previous day, 0.2 µg of mAb antihuman CD61 (IM0540; Beckman Coulter) were used to coat wells (n = 3 wells/condition) by incubating overnight at 4 °C under gentle agitation. Antibodies were removed and unspecific sites were blocked with 300 µl of PBS/BSA 3%/0.05% Tween for 1 hour at RT. Wells were washed three times with 300 µl PBS/0.05% Tween for 5 minutes before addition of 50 µg of protein lysates adjusted to 200 µl with lysis buffer, for 1 hour at RT. Wells were washed again five times with 300 µl PBS/0.05% Tween for 5 minutes. Then, 200 µl of streptavidin-POD/PBS/0.05% Tween (1:10,000) (11 089 153 001; Roche Diagnostic, Meylan, France) were added on anti-CD61/biotinylated integrin complex for 1 hour at RT. Samples were washed three times with 300 µl PBS/0.05% Tween and two times with 300 µl PBS for 5 minutes. At last, integrin internalization was revealed using ABTS kit (00-2011; Zymed, Cergy Pontoise, France) and quantified as described by the manufacturer.

Results were expressed as mean of OD ± SEM and each experiment was performed in quadruplet at least.

Supplementary Material Figure S1. Curve fitting of FCS analysis. Curve fitting of RAFT-RGD-Cy5 mixed with integrin αvβ3 and the corresponding residuals curves using a one-compartment (A) and a two-compartments model (B). Xhi2 values are 5.3E−2 and 1.9E−4, respectively. Using the three-compartments model, the same kinds of curves as in (B) was found, with two similar diffusion times of the complex RAFT-RGD-Cy5/integrin (± 3 μs).

Supplementary Material

Figure S1.

Curve fitting of FCS analysis. Curve fitting of RAFT-RGD-Cy5 mixed with integrin αvβ3 and the corresponding residuals curves using a one-compartment (A) and a two-compartments model (B). Xhi2 values are 5.3E−2 and 1.9E−4, respectively. Using the three-compartments model, the same kinds of curves as in (B) was found, with two similar diffusion times of the complex RAFT-RGD-Cy5/integrin (± 3 μs).

Click here for supplemental data (52.1MB, tiff)

Acknowledgments

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the INCA (Institut National for Cancer), the Association for Research on Cancer (ARC, France), the Agence Nationale pour la Recherche (ANR), and the EMIL and N2L NoE of the 6th FWP. We also acknowledge Marc Block (ERL CNRS 3148, Institut Albert Bonniot BP 170, 38 042 Grenoble Cedex 9, France) for helpful discussion during the redaction of this manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Curve fitting of FCS analysis. Curve fitting of RAFT-RGD-Cy5 mixed with integrin αvβ3 and the corresponding residuals curves using a one-compartment (A) and a two-compartments model (B). Xhi2 values are 5.3E−2 and 1.9E−4, respectively. Using the three-compartments model, the same kinds of curves as in (B) was found, with two similar diffusion times of the complex RAFT-RGD-Cy5/integrin (± 3 μs).

Click here for supplemental data (52.1MB, tiff)

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