Nadir Bettache

Mechanical model of cytoskeleton structuration during cell adhesion and spreading

The biomechanical behavior of an adherent cell is intimately dependent on its cytoskeleton structure. Several models have been proposed to study this structure taking into account its existing internal forces. However, the structural and geometrical complexities of the cytoskeletons filamentous networks lead to difficulties for determining a biologically realistic architecture. The objective of this paper is to present a mechanical model, combined with a numerical method, devoted to the form-finding of the cytoskeleton structure (shape and internal forces) when a cell adheres on a substrate. The cell is modeled as a granular medium, using rigid spheres (grains) corresponding to intracellular cross-linking proteins and distant mechanical interactions to reproduce the cytoskeleton filament internal forces. At the initial state (i.e., before adhesion), these interactions are tacit. The adhesion phenomenon is then simulated by considering microtubules growing from the centrosome towards transmembrane integrin-like receptors. The simulated cell shape changes in this process and results in a mechanically equilibrated structure with traction and compression forces, in interaction with the substrate reactions. This leads to a compressive microtubule network and a corresponding tensile actin-filament network. The results provide coherent shape and forces information for developing a mechanical model of the cytoskeleton structure, which can be exploitable in future biomechanical studies of adherent cells.

Pro-cathepsin D interacts with the extracellular domain of the β chain of LRP1 and promotes LRP1-dependent fibroblast outgrowth

Interactions between cancer cells and fibroblasts are crucial in cancer progression. We have previously shown that the aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer that is overexpressed and highly secreted by breast cancer cells, triggers mouse embryonic fibroblast outgrowth via a paracrine loop. Here, we show the requirement of secreted cath-D for human mammary fibroblast outgrowth using a three-dimensional co-culture assay with breast cancer cells that do or do not secrete pro-cath-D. Interestingly, proteolytically-inactive pro-cath-D remains mitogenic, indicating a mechanism involving protein-protein interaction. We identify the low-density lipoprotein (LDL) receptor-related protein-1, LRP1, as a novel binding partner for pro-cath-D in fibroblasts. Pro-cath-D binds to residues 349–394 of the β chain of LRP1, and is the first ligand of the extracellular domain of LRP1β to be identified. We show that pro-cath-D interacts with LRP1β in cellulo. Interaction occurs at the cell surface, and overexpressed LRP1β directs pro-cath-D to the lipid rafts. Our results reveal that the ability of secreted pro-cath-D to promote human mammary fibroblast outgrowth depends on LRP1 expression, suggesting that pro-cath-D–LRP1β interaction plays a functional role in the outgrowth of fibroblasts. Overall, our findings strongly suggest that pro-cath-D secreted by epithelial cancer cells promotes fibroblast outgrowth in a paracrine LRP1-dependent manner in the breast tumor microenvironment.

Mechanical constraint imposed on plasma membrane through transverse phospholipid imbalance induces reversible actin polymerization via phosphoinositide 3-kinase activation

Platelets were used to explore the effect of membrane curvature induced by phospholipid excess on cell shape and on organization of the actin cytoskeleton. We showed that the addition of short chain analogues of phospholipids to the outer leaflet of plasma membrane of resting platelets immediately induced a shape change with long filopodia formation containing newly polymerized actin. Cells recovered rapidly their discoid shape and their initial F-actin content only with the phosphatidylserine analogue, which was transported to the inner leaflet by aminophospholipid translocase. Filopodia formation and actin polymerization were inhibited in platelets pre-incubated with cytochalasin D. Both wortmannin and LY294002, two unrelated inhibitors of phosphoinositide 3-kinase, considerably reduced actin polymerization and filopodia formation. Phospholipid imbalance was accompanied by a reversible translocation of phosphoinositide 3-kinase from cytoplasm to plasma membrane. In agreement with a role for PI 3-kinase, when phospholipids were added to platelets, PtdIns(3,4)P2 increased two-fold and Akt protein was partly phosphorylated. A similar shape change was also observed in nocodazole-treated L929 fibroblasts which were incubated with the similar phospholipid analogues. In those nucleated cells, where the microtubule cytoskeleton was disrupted, a major actin-dependent membrane extension was induced by addition of short chain phospholipids that required the functional integrity of PI 3-kinase. We conclude that any physical constraint acting on plasma membrane and resulting on local changes in membrane curvature is sufficient to initiate transient actin polymerization via phosphoinositide 3-kinase activation.

Impaired redistribution of aminophospholipids with distinctive cell shape change during Ca2+‐induced activation of platelets from a patient with Scott syndrome

We have investigated phospholipid redistribution, membrane vesicle shedding, shape change, and granule release following A23187 activation of platelets from a patient with Scott syndrome, characterized by impaired transmembrane migration of phosphatidylserine (PS) accompanied by haemorrhagic complications, and two of her children. Electron spin resonance spectroscopy measurement of phospholipids redistribution showed that the internalization of PS was unaffected by the disorder but, after activation, PS exposure was significantly reduced in platelets from the homozygous‐type patient. Vesicle shedding was also reduced in these platelets. However, the slow redistribution of phosphatidylcholine was similar to that observed in normal platelets. When treated with calpeptin, platelets from the homozygous‐type patient, unlike normal or heterozygous Scott syndrome platelets, showed a smoothly rounded shape without filopods after activation. Following A23187 activation of normal platelets, filopod formation was consecutive to the re‐exposition of aminophospholipids on the outer leaflet of the plasma membrane, and the existence of a floppase (outward aminoPLs translocase) has been suggested. In homozygous Scott syndrome platelets the deficiency in PS re‐exposition, the absence of filopod formation, and low vesicle shedding are correlated with each other, and argue in favour of a disruption of the proposed floppase activity.

Structure–Activity Relationships of JMV4463, a Vectorized Cathepsin D Inhibitor with Antiproliferative Properties: The Unique Role of the AMPA-Based Vector

Cathepsin D (CathD) is overexpressed and secreted by several solid tumors and stimulates their growth, the mechanism of which is still not understood. In this context, the pepstatin bioconjugate JMV4463 [Ac‐arg‐O2Oc‐(Val)3‐Sta‐Ala‐Sta‐(AMPA)4‐NH2; O2Oc=8‐amino‐3,6‐dioxaoctanoyl, Sta=statine, AMPA=ortho‐aminomethylphenylacetyl], containing a new kind of cell‐penetrating vector, was previously shown to exhibit potent antiproliferative effects in vitro and to delay the onset of tumors in vivo. In this study, we performed a structure–activity relationship analysis to evaluate the significance of the inhibitor and vector moieties of JMV4463. By modifying both statine residues of pepstatin we found that the antiproliferative activity is correlated with CathD inhibition, supporting a major role of the catalytic activity of intracellular CathD in cancer cell proliferation. Replacing the vector composed of four AMPA units with other vectors was found to abolish cytotoxicity, although all of the conjugates enabled pepstatin transport into cells. In addition, the AMPA4 vector must be localized at the C terminus of the bioconjugate. The unexpected importance of the vector structure and position for cytotoxic action suggests that AMPA4 enables pepstatin to inhibit the proteolysis of critical CathD substrates involved in cell proliferation via a unique mechanism of action.

Ribbon‐like foldamers for cellular uptake and drug delivery

Different intracellular delivery systems of bioactive compounds have been developed, including cell‐penetrating peptides. Although usually nontoxic and biocompatible, these vectors share some of the general drawbacks of peptides, notably low bioavailability and susceptibility to protease degradation, that limit their use. Herein, the conversion of short peptide sequences into poly‐α‐amino‐γ‐lactam foldamers that adopt a ribbon‐like structure is investigated. This template is used to distribute critical cationic and/or hydrophobic groups on both sides of the backbone, leading to potent short, cell‐permeable foldamers with a low positive‐charge content. The lead compound showed dramatically improved protease resistance and was able to efficiently deliver a biologically relevant cargo inside cells. This study provided a simple strategy to convert short peptide sequences into efficient protease‐resistant cell‐penetrating foldamers.

Biomolecular dynamic covalent polymers for DNA complexation and siRNA delivery

Synthetic delivery systems that are described as smart are considered essential for the successful development of gene therapies. Dynamic covalent polymers (DCP) are dynamic and adaptive species that can expand and shorten their main chain in a reversible fashion. In particular, polyacylhydrazone DCPs are pH-sensitive and undergo hydrolytic dissociation at acidic pH, which is an interesting feature for gene delivery. Building upon our previous finding that cationic DCPs can complex DNA through multivalent interactions, we report here on a new generation of DCPs that incorporate modified amino acids. The covalent self-assembly through polycondensation was extended towards multifunctional DCPs combining different building blocks and different molecular dynamics. These biomolecular DCPs were found able to complex both long DNA and siRNA, and biological studies demonstrate that they are able to deliver functional siRNA in living cells. This straightforward and modular approach to the self-production of multifunctional and biomolecular DCPs as siRNA vectors can therefore constitute a stepping stone in smart gene delivery using dynamic and adaptive biodynamers.

Indoloazepinone-Constrained Oligomers as Cell-Penetrating and Blood-Brain-Barrier-Permeating Compounds

Non‐cationic and amphipathic indoloazepinone‐constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l‐Aia‐Xxx]n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc‐[l‐Aia‐Gly]2,4‐OBn oligomers 12 and 13 and Boc‐[l‐Aia‐β3‐h‐l‐Ala]2,4‐OBn oligomers 16 and 17 were totally or partially disordered, Boc‐[l‐Aia‐l‐Ala]2‐OBn (14) induced a typical turn stabilized by C5‐ and C7‐membered H‐bond pseudo‐cycles and aromatic interactions. Boc‐[l‐Aia‐l‐Ala]4‐OBn (15) exhibited a unique structure with remarkable T‐shaped π‐stacking interactions involving the indole rings of the four l‐Aia residues forming a dense hydrophobic cluster. All of the proposed FITC‐6‐Ahx‐[l‐Aia‐Xxx]4‐NH2 oligomers 19–23, with the exception of FITC‐6‐Ahx‐[l‐Aia‐Gly]4‐NH2 (18), were internalized by MDA‐MB‐231 cells with higher efficiency than the positive references penetratin and Arg8. In parallel, the compounds of this series were successfully explored in an in vitro blood–brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac‐[l‐Aia‐Xxx]4‐NH2 oligomers in the PAMPA model, Ac‐[l‐Aia‐l‐Arg]4‐NH2 (26) showed significant permeation in the in vitro cell‐based human model of the BBB, suggesting an active mechanism of cell penetration.

NUMERICAL MODEL OF THE CYTOSKELETON STRUCTURATION DURING CELL SPREADING

The objective of this work is to propose a mechanical model and a numerical method devoted to the cytoskeleton (CSK) form-finding resulting from its structuration during cell spreading. It is now well assumed that the cell mechanics closely depends on its CSK architecture and on the tension and compression forces carried by its filaments. Several structural models have been therefore developed, especially those based on the tensegrity analogy. However, the observed topological and geometrical complexities of the CSK networks lead to difficulties for determining and using a realistic structure [Baudriller et al., 2006]. The presented method allows calculating such a complex architecture and the associated forces in the different CSK filaments of an adherent cell.

Phospholipids in Platelets: Localization, Movement and Physiological Function

Hemostasis is the control of blood circulation in vessels by a very complex and fast cascade of proteolytic reactions. In the case of vascular lesions, a major activator complex is formed between tissue factor (TF) and factor VII. At the same time, platelets adhering to subendothelial components secrete their granule contents and activate other platelets in a chain reaction. These platelets aggregate in turn while their plasma membrane rapidly becomes able to bind coagulating factors (VIIIa-IXa in tenase complex; VA-Xa in prothrombinase complex), essentially through their phosphatidylserine (PS) outer surface content. Finally, the procoagulant power of PS-containing membranes depends on their ability to assemble tenase and prothrombinase complexes and to protect activated factors against endogenous anticoagulating factors (Mann et al., 1990).