Biology subjects

Cribier, S.

Publications and source records attributed to Cribier, S..

3 recordsLinked to original sources

bioRxiv2026

Covalently linked peptides and membrane potential enable CyaA segment translocation

The adenylate cyclase toxin (CyaA) from Bordetella pertussis intoxicates host cells by directly translocating its N-terminal catalytic domain across the plasma membrane; however, the forces driving this unique process remain poorly defined. Here, we dissect the membrane translocation mechanisms of two peptide segments derived from CyaA: P233 and P454 from the catalytic domain and the translocation region, respectively. Both P454 and P233 are calmodulin-binding segments that are sequentially involved in the translocation and activation of the catalytic domain. Using a newly developed Droplet Interface Bilayer (DIB) approach, called DIB-Pipette, which enables direct visualization of peptide transport under controlled membrane potentials, we show that P454 translocates across membranes independently of membrane potential, whereas P233 translocation requires a negative electric membrane potential. Strikingly, covalent coupling of P233 and P454 enables efficient translocation of the resulting peptide even in the absence of a membrane potential. Together, these results suggest that two distinct membrane-active segments within CyaA act cooperatively to promote translocation at the peptide level, revealing an intrinsic mechanism that may contribute to membrane potential-dependent translocation. These findings provide new mechanistic insights into CyaA cell intoxication process and reveal a multifunctional strategy for protein delivery across membranes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/716334v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@750ab3org.highwire.dtl.DTLVardef@11a980org.highwire.dtl.DTLVardef@18f2b27org.highwire.dtl.DTLVardef@5a3a59_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Asymmetric phosphoinositide lipid bilayers generated by spontaneous lipid insertion

Phosphatidylinositol phosphate (PIP) lipids, enriched on the cytoplasmic leaflet of the plasma membrane, are key regulators of diverse cellular processes, often through interactions with partner proteins that regulate actin assembly. Supported lipid bilayers (SLBs) provide a powerful model system to study the interactions of PIP lipids with their partner proteins. However, despite advances in SLB preparation methods, it remains a challenge to robustly obtain fluid SLBs in which PIP lipids are both mobile and asymmetrically distributed. In this study, we report a simple and robust method to generate asymmetric SLBs containing tunable amounts of PI(4,5)P2. By dissolving PI(4,5)P2 below its critical micelle concentration (CMC), we enable its spontaneous insertion into the upper leaflet of SLBs exposed to bulk solution. The mobility of PI(4,5)P2 is confirmed using fluorescence recovery after photobleaching (FRAP). Furthermore, we demonstrate that PI(4,5)P2 incorporated using this method retains its functionality, recruiting binding partners, actin-membrane linker ezrin, and myosin 1 motors capable of sliding actin filaments on the SLBs. Our method offers a straightforward strategy to generate asymmetric PI(4,5)P2-containing SLBs and is applicable to other lipid species with high CMC values.

biophysics↗

bioRxiv2020

Specific interaction of Penetratin with cell surface partners measured with biomembrane force probe

Penetratin is a Cell Penetrating Peptide able to cross the cell plasma membrane possibly bound to a cargo molecule to be delivered into the cell. The mechanism of its entry is poorly known. A key to a molecular description of this mechanism is to identify the partners of Penetratin at the cell surface during its adhesion and internalization. We used the Biomembrane Force Probe to identify the partners during the first second of adhesion of Penetratin on the cell plasma membrane. We evidenced that heparan sulfates are the first partners after contact as well as unknown partners hidden by sialic acids. Experiments of binding of Penetratin on vesicles bearing charged or sulfated lipids showed no adhesion pointing that a negatively charged partner is not enough and there is a specificity for certain chemical groups bearing the charges. A model of the measured forces of interaction enabled to determine the adhesion energy of a Penetratin with heparan sulfates on a cell to be in the range 18 to 22 kBT.

biophysics↗