Biomedical subjects

Pierre-Emmanuel Milhiet

Publications and source records attributed to Pierre-Emmanuel Milhiet.

6 recordsLinked to original sources

PubMed2006

High-resolution AFM of membrane proteins directly incorporated at high density in planar lipid bilayer.

The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl-beta-maltoside or dodecyl-beta-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20 degrees C and at 4 degrees C with three bacterial photosynthetic multi-subunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of two-dimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.

Elasticity↗

PubMed2005

Coexistence of a two-states organization for a cell-penetrating peptide in lipid bilayer.

Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P(alpha), a primary amphipathic cell-penetrating peptide which remains alpha-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P(alpha) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P(alpha) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P(alpha). The simultaneous two-states organization of P(alpha), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.

Lipid Bilayers↗

PubMed2004

Calcitonin-derived carrier peptide plays a major role in the membrane localization of a peptide-cargo complex.

Bilayers made of dioleoylphosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC) mixture containing or not cholesterol (Chl) were used to investigate the interaction of a carrier peptide with membranes. Atomic force microscopy revealed that the C-terminal 9-32 fragment of human calcitonin (hCT (9-32)), free or coupled to enhanced green fluorescent protein (hCT-eGFP) cargo forms aggregates in the DOPC fluid phase in absence of Chl and in the DPPC enriched liquid-ordered phase when Chl is present. The data show that hCT (9-32) plays a determinant role in the membrane localization of the peptide-cargo complex. They suggest that carpet-like mechanism for membrane destabilization may be involved in the carrier function of hCT (9-32).

Amino Acid Sequence↗

PubMed2004

Heparin affin regulatory peptide in milk: its involvement in mammary gland homeostasis.

HARP (heparin affin regulatory peptide) is a heparin binding growth factor implicated in cellular growth and differentiation. Previously, HARP had been localized in the human mammary, in both alveolar epithelial and myoepithelial cells although HARP mRNAs were only expressed by myoepithelial cells [J. Histochem. Cytochem. 45 (1997) 1]. In the present study, we demonstrate that HARP is secreted in human mature milk with concentrations ranging from 17.68+/-6.4ng/ml in mature milk to 59.9+/-11.22ng/ml in colostrum. In vitro, HARP was found to be mitogenic on human mammary epithelial and myoepithelial cell lines and correlated with the expression of its high affinity receptor tyrosine kinase ALK (anaplastic lymphoma kinase). In vivo, ALK is expressed in both mammary epithelial and myoepithelial cells, suggesting that HARP could act in vivo as a paracrine and autocrine growth factor in the regulation of the mammary gland development and its homeostatic maintenance during pregnancy and lactation.

Anaplastic Lymphoma Kinase↗

PubMed2004

[Atomic force microscopy: from cell imaging to molecular manipulation].

The atomic force microscope (AFM) allows to explore the surface of biological samples bathed in physiological solutions, with vertical and horizontal resolutions ranging from nanometers to angströms. Complex biological structures as well as single molecules can be observed and recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra and intermolecular forces from single molecules are also presented.

Animals↗

PubMed2002

Spontaneous insertion and partitioning of alkaline phosphatase into model lipid rafts.

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C-terminal end that serves as an anchor to the plasma membrane and could be responsible for the presence of GPI proteins in rafts, a type of functionally important membrane microdomain enriched in sphingolipids and cholesterol. In order to understand better how GPI proteins partition into rafts, the insertion of the GPI-anchored alkaline phosphatase (AP) was studied in real-time using atomic force microscopy. Supported phospholipid bilayers made of a mixture of sphingomyelin-dioleoylphosphatidylcholine containing cholesterol (Chl+) or not (Chl-) were used to mimic the fluid-ordered lipid phase separation in biological membranes. Spontaneous insertion of AP through its GPI anchor was observed inside both Chl+ and Chl- lipid ordered domains, but AP insertion was markedly increased by the presence of cholesterol.

Alkaline Phosphatase↗