ARTICLE Received 18 Apr 2014 | Accepted 2 Aug 2014 | Published 3 Oct 2014 DOI: 10.1038/ncomms5904 An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability Hong-Bo Pang1, Gary B. Braun1,2, Tomas Friman1,2, Pedro Aza-Blanc1, Manuel E. Ruidiaz1, Kazuki N. Sugahara1,3, Tambet Teesalu1,4 & Erkki Ruoslahti1,2 Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signalling molecules bind to NRPs through a carboxy (C)-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumour-homing CendR peptides penetrate through tumour tissue and have shown utility in enhancing drug delivery into tumours. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signalling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway. 1 Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA. 2 Center for Nanomedicine, Department of Cell, Molecular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California 93106-9610, USA. 3 Department of Surgery, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA. 4 Laboratory of Cancer Biology, Institute of Biomedicine and Translational Medicine, Centre of Excellence for Translational Medicine, University of Tartu, 50411 Tartu, Estonia. Correspondence and requests for materials should be addressed to E.R. (email: [email protected]). NATURE COMMUNICATIONS | 5:4904 | DOI: 10.1038/ncomms5904 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5904 ransport of molecules across the vascular wall, through appear in endocytic vesicles inside the cells4. The cytoplasmic tissue and into target cells have an important role in domain of NRP1 contains a PSD-95/Dlg/ZO-1 (PDZ) binding Tvarious physiological and pathological processes1,2.Itis motif (SEA-COOH), which interacts with the PDZ domain of the also a major limiting factor in drug delivery, particularly into cytoplasmic protein GIPC1/synectin6. The NRP1–GIPC1/ extravascular tumour tissue3. We recently identified a class of synectin interaction is known to have a role in the trafficking peptides that were particularly effective in entering into cultured of endocytosed VEGFR2 into Rab5a-positive endosomes upon 12 cells and that the peptides and cargo attached to them appeared VEGF-A165 stimulation . in endocytic vesicles inside the cells4. These peptides shared a Several types of endocytosis have been described21–23.Best carboxy (C)-terminal consensus motif, R/KXXR/K, and we understood is the molecular machinery of clathrin-mediated showed that the prototype peptide among them, RPARPAR, endocytosis (CME), which is responsible for most of the binds to neuropilin-1 (NRP1) on the target cells4. receptor-mediated uptake into cells24. Caveolae-mediated NRP1 is a cell surface receptor with multiple ligands that bind endocytosis (Cav-ME) utilizes plasma membrane invaginations to NRP1 through a C-terminal R/KXXR/K motif similar to known as caveolae, which are formed as a result of caveolin RPARPAR. These ligands include vascular endothelial growth (CAV) oligomerization and are enriched in cholesterol23. factor (VEGF)-A isoform, VEGF-A165, transforming growth Macropinocytosis (MP) functions to nonselectively engulf 5–7 factor b and semaphorin 3A (Sema3A) . VEGF-A165 and extracellular solute macromolecules into endosomes in a Sema3A are known to increase vascular permeability to receptor-independent manner22. There are no known molecular macromolecules8,9. These complex molecules have additional markers for macropinosomes, but a characteristic feature of these receptors to trigger downstream signalling events, but the vesicles is their heterogeneous size (0.2–5 mm in diameter), while vascular permeability effect is mediated by NRP1 (ref. 10), clathrin-coated vesicles and caveolae are usually more despite the fact that NRP1 lacks an intracellular signalling homogeneous, spherical in shape, and much smaller (85–150 nm domain6. Thus, the RPARPAR peptide, and a peptide from the C diameter)22. Given the apparent importance of the CendR pathway 4 terminus of VEGF-A165, both trigger vascular permeability . in receptor trafficking and its potential in drug delivery, we set out The increased vascular permeability obtained by NRP1 ligation to study the molecular machinery that mediates and regulates may be based on extravasation through a paracellular or CendR-mediated cell and tissue penetration. Results of genome- transcellular route, or both. A peptide modelled after the C wide RNA interference (RNAi) screens, together with mechanistic terminus of Sema3A has been reported to loosen cell junctions in and morphological studies of CendR-mediated cell entry and the endothelium, which is thought to be responsible for the subsequent intercellular transport show that the CendR pathway is increased vascular permeability triggered by this peptide11. distinct from known endocytic pathways, and that this pathway is However, NRP1 is also involved in endocytosis. It initiates regulated by nutrient supply to cells and tissues. intracellular trafficking of a number of membrane receptors and regulates the transport of integrins from one part of a cell to another12,13. Moreover, Dvorak and co-workers showed that Results endothelial cells treated with VEGF-A164 (mouse orthologue of Genome-wide RNAi screen for CendR-mediated cell entry.To human VEGF-A165) developed striking assemblies of intracellular study cell entry of NRP1-binding peptides, we monitored the vesicles that they named as vesiculo–vacuolar system and internalization of RPARPAR into a prostate tumour cell line postulated to be involved in transcytosis14,15. However, the (PPC1). PPC1 cells were chosen for this study because these cells ability of NRP1-binding peptides to induce endocytosis and tissue are particularly efficient in internalizing CendR peptides4. penetration has generally not been appreciated. RPARPAR was identified in a phage display screen as the Small peptides with a C-terminal R/KXXR/K motif are useful in strongest binder to these NRP1-expressing cells4. The CendR investigating the functions of NRP1 because unlike the natural motif of RPARPAR is required for peptide binding to NRP1 and ligands of NRP1, which usually bind to additional receptors, the internalization into cells4. After entry, RPARPAR colocalizes with peptides bind only to NRP1, and only to a single site in it. To endosomal and lysosomal markers inside cells4. achieve NRP1 binding and initiate the subsequent endocytosis and We performed a genome-wide RNAi screen to isolate genes tissue penetration, a peptide must have R/KXXR/K at their free C that regulate the cell entry of CendR peptides (CendR terminus4. Hence, we have named the system the C-end Rule endocytosis, Fig. 1a). To create a probe for the screen, we coated (CendR) pathway and the R/KXXR/K motif the CendR motif4. RPARPAR onto fluorescently labelled silver nanoparticles (R-Ag, NRP1 is ubiquitously expressed in the vasculature and many 70±10 nm, ref. 25). The silver nanoparticles greatly enhance the other cell types. However, a class of tumour-homing peptides that emission from fluorescent dyes coupled to their surface. Another specifically activate the pathway in tumours has been described advantage of this silver-based platform is that it allows exclusive recently3. These peptides contain a CendR motif rendered cryptic tracing of the internalized fraction of R-Ag because the by an internal position in the peptide. Each of them uses a different extracellular R-Ag can be readily removed by employing a mild receptor for initial homing to the target tissue, but converges on etching solution25. The fluorescence intensity of R-Ag NRP1 after a proteolytic processing step at the surface of the target internalized into cells remained stable for up to 24 h cells that activates the cryptic CendR motif3,16,17.Thesetumour- (Supplementary Fig. 1A). Treatment with NRP1 siRNA (small specific CendR peptides are capable of penetrating tumour vascular interfering RNA, positive control) efficiently blocked R-Ag walls and transporting molecular and nanoparticle payloads attached uptake, similar to NRP1 blocking antibody, whereas nonspecific to the peptide into and through extravascular tumour tissue4,16,18,19. (NS) siRNAs had no obvious effect (Supplementary Fig. 1B). The Remarkably, CendR pathway also transports cargo that is nearby but NRP1 knockdown was verified using quantitative PCR (qPCR) not chemically conjugated to the peptide (bystander effect), making and immunoblotting (Supplementary Fig. 1C). To validate the it possible to enhance drug delivery to tumours by co-administering quality and to evaluate the reproducibility of the assay, we also the peptide with an unmodified drug19. performed a mini screen using 350 randomly