A Genome-Wide Collection of Mos1 Transposon Insertion Mutants for the C. elegans Research Community

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource

FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse

Axonal receptors for class 3 semaphorins (Sema3s) are heterocomplexes of neuropilins (Nrps) and Plexin-As signalling coreceptors. In the developing cerebral cortex, the Ig superfamily cell adhesion molecule L1 associates with Nrp1. Intriguingly, the genetic removal of L1 blocks axon responses of cortical neurons to Sema3A in vitro despite the expression of Plexin-As in the cortex, suggesting either that L1 substitutes for Plexin-As or that L1 and Plexin-A are both required and mediate distinct roles. We report that association of Nrp1 with L1 but not Plexin-As mediates the recruitment and activation of a Sema3A-induced focal adhesion kinase-mitogen-activated protein kinase cascade. This signalling downstream of L1 is needed for the disassembly of adherent points formed in growth cones and subsequently their collapse response to Sema3A. Plexin-As and L1 are coexpressed and present in common complexes in cortical neurons and both dominant-negative forms of Plexin-A and L1 impair their response to Sema3A. Consistently, Nrp1-expressing cortical projections are defective in mice lacking Plexin-A3, Plexin-A4 or L1. This reveals that specific signalling activities downstream of L1 and Plexin-As cooperate for mediating the axon guidance effects of Sema3A

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

International audienceInsufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH41 1 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or Idlr-!-mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells-including pathological cells-or organs that express the LDLR

Chemical optimization of new ligands of the low-density lipoprotein receptor as potential vectors for central nervous system targeting.

International audienceDrug delivery to the central nervous system is hindered by the presence of physiological barriers such as the blood-brain barrier. To accomplish the task of nutrient transport, the brain endothelium is endowed with various transport systems, including receptor-mediated transcytosis (RMT). This system can be used to shuttle therapeutics into the central nervous system (CNS) in a noninvasive manner. Therefore, the low-density lipoprotein receptor (LDLR) is a relevant target for delivering drugs. From an initial phage display biopanning, a series of peptide ligands for the LDLR was optimized leading to size reduction and improved receptor binding affinity with the identification of peptide 22 and its analogues. Further real-time biphoton microscopy experiments on living mice demonstrated the ability of peptide 22 to efficiently and quickly cross CNS physiological barriers. This validation of peptide 22 led us to explore its binding on the extracellular LDLR domain from an NMR-oriented structural study and docking experiments

A midline switch of receptor processing regulates commissural axon guidance in vertebrates

Commissural axon guidance requires complex modulations of growth cone sensitivity to midline-derived cues, but underlying mechanisms in vertebrates remain largely unknown. By using combinations of ex vivo and in vivo approaches, we uncovered a molecular pathway controlling the gain of response to a midline repellent, Semaphorin3B (Sema3B). First, we provide evidence that Semaphorin3B/Plexin-A1 signaling participates in the guidance of commissural projections at the vertebrate ventral midline. Second, we show that, at the precrossing stage, commissural neurons synthesize the Neuropilin-2 and Plexin-A1 Semaphorin3B receptor subunits, but Plexin-A1 expression is prevented by a calpain1-mediated processing, resulting in silencing commissural responsiveness. Third, we report that, during floor plate (FP) in-growth, calpain1 activity is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone and sensitization to Sema3B. Finally, we show that the FP cue NrCAM mediates the switch of Plexin-A1 processing underlying growth cone sensitization to Sema3B. This reveals pathway-dependent modulation of guidance receptor processing as a novel mechanism for regulating guidance decisions at intermediate targets

Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor

International audienceActive targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC]c), which showed the highest KD value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 10(6) s(-1.)M(-1) range, and off-rates varying from the low 10(-2) s(-1) to the 10(-1) s(-1) range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"]c), showing a KD of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled (3)H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo

Binding of (VH434)₂-Fc, (VH4127)₂-Fc, Fc and (VH4Sc)₂-Fc to the hLDLR.

<p>Representative confocal fluorescence micrograph of CHO-hLDLR-GFP cells incubated with 10 nM of (VH434)₂-Fc (a), (VH4127)₂-Fc (b) and Fc alone (c) used as a negative control. The Fc and Fc-fusion/conjugates were detected with an anti-hFc Alexa 594-conjugated antibody (red). Co-labeling of LDLR-GFP (green) and Fc fusion/conjugates appear in yellow in the merged pictures, as evidenced at higher magnification. (B) ELISA quantification of bound/endocytosed (VH4127)₂-Fc or (VH4Sc)₂-Fc conjugates to CHO-hLDLR-GFP cells 1 hr post-incubation at 10 nM (n = 3 per conjugate; ***p ≤ 0.001). Note that (VH4127)₂-Fc binding/endocytosis to the CHO-hLDLR-GFP cells is increased ≈300-fold compared to the (VH4Sc)₂-Fc control. (C) Representative sensorgrams of Fc-fusion/conjugate binding to the extracellular domain of hLDLR. A set of concentrations (0.5–80 nM for the Fc-conjugates or 5–80 nM for the Fc-fusion) was sequentially injected over immobilized hLDLR. The solid lines represent the specific binding of each Fc-fusion/conjugate and the dotted lines represent the fit of the data obtained with a Langmuir 1:1 model.</p

Sequences of RT-PCR primers.

<p>Sequences of RT-PCR primers.</p

<i>In vitro</i> validation of VH411 phage binding.

<p>(A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10¹⁰ VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.</p