Jesus Ayala-Sanmartin

• Institute of Biological Sciences, French National Centre for Scientific Research

Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)...

Cell penetrating peptides (CPPs) are able to transport hydrophilic molecules inside cells. To reach the cytosol, the peptide associated with a cargo must cross the plasma or the endosomal membrane. Different molecular mechanisms for peptide internalisation into cells have been proposed and it is becoming clear that the cellular internalisation mech...

CPPs effect on cholesterol-pyrene multimers in function of temperature on SM/Chol LUVs. The excimers/isoemisive ratio (474/432 nm) was followed at different temperatures during heating (A), or cooling (B) of the samples. CPPs were incubated with the LUVs at a 1/25 P/L ratio. Control CPP free LUVs black, Penetratin green, R9 blue and RW9 red. Means...

CPPs effect on cholesterol-pyrene multimers in function of temperature on PC/SM/Chol LUVs. The excimers/isoemisive ratio (474/432 nm) was followed at different temperatures during heating (A), or cooling (B) of the samples. CPPs were incubated with the LUVs at a 1/25 P/L ratio. At a 1/10 P/L ratio the peptides effects on excimers were identical to...

CPPs effects on cholesterol-pyrene liquid disordered environment in function of temperature on PC/SM/Chol LUVs. The different ratios were followed at 379/432 temperatures during heating (continuous lines), or cooling (dotted lines) of the samples. CPPs were incubated with the LUVs at a 1/25 P/L ratio (top panels) and 1/10 ratio (bottom panels). The...

CCPs internalisation into LUVs. (A) Protocol for time-dependent percent of total fluorescence evolution. LUVs were incubated with NBD-labelled CPPs. Dithionite is added to quench free in solution CPPs. After stabilization, melittin was added allowing the quenching of CPP inside the LUVs lumen. Therefore, the difference of fluorescence before and af...

CPPs effects on cholesterol-pyrene fluorescence liquid disordered environment in function of temperature on SM/Chol LUVs. The 379/432 ratios were followed at different temperatures during heating (continuous lines), or cooling (dotted lines) of the samples. CPPs were incubated with the LUVs at a 1/25 P/L ratio (top panels) and 1/10 ratio (bottom pa...

CPPs effect on cholesterol-pyrene multimers in function of temperature on PC LUVs. The excimer/iso-emissive ratio (474/432 nm) was followed at different temperatures during heating (A), or cooling (B). CPPs were incubated with the LUVs at a 1/10 P/L ratio. Control CPP free LUVs (black ●,○), Penetratin (green ♦,◊), R9 (blue ▲,∆) and RW9 (red ■,□). M...

CPPs effects on cholesterol-pyrene fluorescence parameters (cholesterol environment) in function of temperature on PC LUVs. The different ratios were followed at different temperatures during heating (continuous lines), or cooling (dotted lines) of the samples. CPPs were incubated with the LUVs at a 1/25 P/L ratio (top panels) and 1/10 ratio (botto...

Biological membranes contain a large variety of lipids species compartmentalized in different domains heterogeneous in size, composition and dynamics. Cholesterol induces membrane ordered domains thanks to its affinity for saturated lipids. Membrane domains had been studied with fluorescent probes either linked to phospholipids and proteins or as i...

Ratios of Py-met-chol fluorescence in mixed membranes. The probe concentration in LUVs is 1.8%. (A), cPyD9/iso during heating. (B), cPyD9/iso during cooling. (C), cPyO3/iso during heating. (D), cPyO3/iso during cooling. (E),exci/iso during heating. (F), exci/iso during cooling. Black lines PC LUVs, red lines SM/Chol and green lines PC/SM/Chol. Cont...

Mean spectra of PC and SM/Chol LUVs at different temperatures after cooling and heating. (A) spectra at 10°C, (B) at 35°C and (C) at 55°C. Red lines PC LUVs, black lines SM/Chol LUVs. Continuous lines from heating protocol and dotted lines from cooling protocol. Each line is the mean of 7 (PC) and 8 (SM/Chol) spectra from independent experiments. (...

Ratios of Py-met-chol fluorescence wavelengths in function of temperature. The probe concentration in LUVs is 1.8%. (A), cPyD9/iso. (B), cPyO3/iso. (C), I1/I3 (“polarity/constant”). (D), pol/iso. (E), exci/I3. (F), exci/iso. Black lines PC LUVs, red lines SM/Chol. Continuous lines heating protocol and dotted lines cooling protocol. Means ± SEM of 4...

Fluorescence anisotropy (r) of Py-met-chol. LUVs were incubated at (A) 15°C, (B) 50°C. Py-met-chol is at 3.6%, PC LUVs in black and SM/Chol in red. Curves are the mean ± SEM of four independent experiments. (JPG)

Ceramide effect on the evolution of Py-met-chol fluorescence in function of temperature. The probe concentration in LUVs is 3.6%. (A), cPyD9 marker. (B), cPyO3 marker. Green lines (▲,∆) PC/SM/Chol LUVs, blue lines (♦,◊) PC/SM/Cer/Chol LUVs. Continuous lines heating protocol and dotted lines cooling protocol. Means ± SEM of 3 independent experiments...

Evolution of Py-met-chol fluorescence intensities in function of temperature. The probe concentration in LUVs is 1.8%. (A), 379 nm Ld marker (cPyD9). (B), 373 nm Lo marker (cPyO3). (C), 388 nm I3 “constant pic”. (D), 432 nm iso-emissive “constant point”. (E), 376 nm I1 “polarity pic”. (F), 401 nm “polarity”. (G), 474 nm excimer pic or multimer mark...

cPyO3/cPyD9 ratios of Py-met-chol fluorescence as function of temperature. The probe concentration in LUVs is 1.8%. Black lines PC LUVs, red lines SM/Chol and green lines PC/SM/Chol. Continuous lines heating protocol and dotted lines cooling protocol. Means ± SEM of 4 independent experiments for PC, 5 for SM/Chol, and 6 for PC/SM/Chol. (JPG)

Subtraction of Ld-Lo Py-met-chol spectra (3.6%) at different temperatures. PC mean spectrum minus SM/Chol mean spectrum at (A) 10°C, (B) 35°C and (C) 55°C. Continuous lines for heating protocol and dotted line for cooling protocol. Mean of 7 independent experiments for PC and 8 for SM/Chol. (JPG)

Multivariate analysis of experimental individuals (spectra) and variables (wavelength). (A, B) Principal component analysis. (A), Distribution of data in a plane of axis corresponding to the two principal components (score scatter plot). Each point represents a spectrum from cooling and heating protocols (300 spectra). In dark blue points PC during...

Cell penetrating peptides are promising vectors for molecular drug delivery in eukaryotic cells. Despite of their discovery 20 years ago, the mechanisms of peptide membrane crossing are still controversial. The different suggested penetration mechanisms reflect the high sequence and structural diversity of cell penetrating peptides. The fundamental...

Annexin A2 (AnxA2) is a phospholipid binding protein that has been implicated in many membrane-related cellular functions. AnxA2 is able to bind different acidic phospholipids such as phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI2P). This binding is mediated by Ca(2+)-dependent and Ca(2+)-independent mechanisms. The specific...

EPEC (enteropathogenic Escherichia coli) and EHEC (enterohaemorrhagic Escherichia coli) are attaching and effacing pathogens frequently associated with infectious diarrhoea. EPEC and EHEC use a T3SS (type III secretion system) to translocate effectors that subvert different cellular processes to sustain colonization and multiplication. The eukaryot...

Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial...

Protein membrane transduction domains are able to translocate through cell membranes. This capacity resulted in new concepts on cell communication and in the design of vectors for internalization of active molecules into cells. Penetratin crosses the plasma membrane by a receptor and metabolic energy-independent mechanism which is at present unknow...

Annexin 2, a member of the annexin family of Ca2+-dependent membrane binding proteins is found in monomeric and heterotetrameric forms and has been involved in different membrane related functions. The heterotetrameric annexin 2 is composed of a dimer of S100A10, a member of the S100 family of Ca2+ binding proteins and two annexin 2 molecules ((Anx...

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expressi...

The delivery of active molecules into cells requires the efficient translocation of the plasma membrane barrier. Penetratin is a promising cell penetrating peptide is which crosses the cell membrane by a receptor and metabolic energy-independent mechanism. In previous work, we have shown that basic peptides induce membrane invaginations (i.e., tube...

Dermaseptin B2 (Drs B2) is a 33-residue-long cationic, alpha-helical antimicrobial peptide endowed with membrane-damaging activity against a broad spectrum of microorganisms, including bacteria, yeasts, fungi, and protozoa, but its precise mechanism of action remained ill-defined. A detailed characterization of peptide-membrane interactions of Drs...

Annexin A2 (AnxA2) is a Ca(2+)- and acidic phospholipid-binding protein involved in many cellular processes. It undergoes Ca(2+)-mediated membrane bridging at neutral pH and has been demonstrated to be involved in an H(+)-mediated mechanism leading to a novel AnxA2-membrane complex structure. We used fluorescence techniques to characterize this H(+...

Annexin A2 (AnxA2) is a Ca(2+)- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca(2+) binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth o...

Background: Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating p...

Fine tubes formed in GUVs by R9 peptide. (1.06 MB MOV)

Start of a growing tube induced by pAntp. (1.62 MB MOV)

Growing tubes induced by RW16 peptide. (0.99 MB MOV)

Annexin 2-GFP movement from cytosol to the plasma membrane induced by (0.42 MB MOV)

pAntp induced adhesion of GUVs. (0.19 MB MOV)

Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and ar...

Cytoplasmic phospholipase A2 (cPLA2) has a key role in prostaglandin production. The role of cPLA2 in intestinal tumorigenesis has been suggested, however, contradictory data are found in the literature. We evaluated cPLA2 and cyclooxygenase-2 (COX-2) protein expression in 65 colon carcinomas by immunohistochemistry, and in eight colorectal cancer...

Peptide-membrane interaction is the first step required for peptide cell internalization. In this paper we studied the interactions of substance P, Penetratin and an amphiphilic 16mer (RL16) peptide in two different model membranes, giant unilamellar vesicles and large unilamellar vesicles. Penetratin was able to induce the formation of tubes insid...

Annexin 2 is a Ca2+-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitoge...

Annexin 2 belongs to the annexin family of proteins that bind to phospholipid membranes in a Ca2+-dependent manner. Here we show that, under mild acidic conditions, annexin 2 binds to and aggregates membranes containing anionic phospholipids, a fact that questions the mechanism of its interaction with membranes via Ca2+ bridges only. The H+ sensiti...

Annexin II heavy chain (also called p36, calpactin I) is lost in prostate cancers and in a majority of prostate intraepithelial neoplasia (PIN). Loss of annexin II heavy chain appears to be specific for prostate cancer since overexpression of annexin II is observed in a majority of human cancers, including pancreatic cancer, breast cancer and brain...

Annexin 2 is a Ca(2+)-dependent phospholipid-binding protein that is involved in secretion. Despite the fact that this protein does not have signals for its secretion, many reports have shown its presence in the extracellular milieu. Here we demonstrate that, upon stimulation of exocytosis in chromaffin cells, a fraction of annexin 2 is secreted in...

Annexins are Ca(2+)-dependent phospholipid-binding proteins composed of two domains: A conserved core that is responsible for Ca(2+)- and phospholipid-binding, and a variable N-terminal tail. A Ca(2+)-independent annexin 2-membrane association has been shown to be modulated by the presence of cholesterol in the membranes. Herein, the roles of the c...

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released b...

The modulation of the local structure and dynamics of domain III of annexin 2 (Anx2), in both the monomeric (p36) and heterotetrameric forms (p90), by calcium and by membrane binding was studied by time-resolved fluorescence intensity and anisotropy measurements of the single tryptophan residue (W212). The results yield the same dominant excited-st...

Annexin 2 binds and aggregates biological membranes in a Ca(2+)-dependent manner. This protein exists as a monomer (p36) or as a heterotetramer (p90) in which two p36 chains are associated with a dimer of p11, a member of the S100 protein family. Protein kinase C phosphorylates the protein at the level of the N-terminal tail on serines 11 and 25, t...

Chimeric polypeptides composed of the homeodomain of Antennapedia and of the C-terminus of several low molecular weight GTP-binding proteins of the rab family have been found to translocate through the membrane of cells in culture and to accumulate in the cytoplasm and nucleus. We have used these chimeric peptides to study, in intact endocrine cell...

The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1‐like and pBR322‐like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid sta...

The role of tumour necrosis factor (TNF-α) in brain physiology and pathology has been the focus of several studies. However, the source of this lymphokine in the central nervous system and the regulation of its synthesis is still poorly understood. We have therefore used purified astrocytes and brain macrophages in culture to compare the abilities...