Yaya Lefkir

Ent3p Is a PtdIns(3,5)P2 Effector Required for Protein Sorting to the Multivesicular Body

PtdIns(3,5)P(2) is required for cargo-selective sorting to the vacuolar lumen via the multivesicular body (MVB). Here we show that Ent3p, a yeast epsin N-terminal homology (ENTH) domain-containing protein, is a specific PtdIns(3,5)P(2) effector localized to endosomes. The ENTH domain of Ent3p is essential for its PtdIns(3,5)P(2) binding activity and for its membrane interaction in vitro and in vivo. Ent3p is required for protein sorting into the MVB but not for the internalization step of endocytosis. Ent3p is associated with clathrin and is necessary for normal actin cytoskeleton organization. Our results show that Ent3p is required for protein sorting into intralumenal vesicles of the MVB through PtdIns(3,5)P(2) binding via its ENTH domain.

New COP1‐binding motifs involved in ER retrieval

Coatomer‐mediated sorting of proteins is based on the physical interaction between coatomer (COP1) and targeting motifs found in the cytoplasmic domains of membrane proteins. For example, binding of COP1 to dilysine (KKXX) motifs induces specific retrieval of tagged proteins from the Golgi back to the endoplasmic reticulum (ER). Making use of the two‐hybrid system, we characterized a new sequence (δL) which interacts specifically with the δ‐COP subunit of the COP1 complex. Transfer of δL to the cytoplasmic domain of a reporter membrane protein resulted in its localization in the ER, in yeast and mammalian cells. This was due to continuous retrieval of tagged proteins from the Golgi back to the ER, in a manner similar to the ER retrieval of KKXX‐tagged proteins. Extensive mutagenesis of δL identified an aromatic residue as a critical determinant of the interaction with COP1. Similar COP1‐binding motifs containing an essential aromatic residue were identified in the cytoplasmic domain of an ER‐resident protein, Sec71p, and in an ER retention motif previously characterized in the CD3ϵ chain of the T‐cell receptor. These results emphasize the role of the COP1 complex in retrograde Golgi‐to‐ER transport and highlight its functional similarity with clathrin–adaptor complexes.

The ADP-ribosylation factor GTPase-activating protein GIo3p is involved in ER retrieval

Retrograde transport of proteins from the Golgi to the endoplasmic reticulum (ER) has been the subject of some interest in the recent past. Here a new thermosensitive yeast mutant defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum was characterized. The ret4-1 mutant also exhibited a selective defect in forward ER-to-Golgi transport of some secreted proteins at the non-permissive temperature. The corresponding RET4 gene was found to encode Glo3p, a GTPase-activating protein (GAP) specific for ADP-ribosylation factor (ARF). In vitro, the Glo3 thermosensitive mutant showed a reduced ARF1-GAP activity. The Glo3 protein belongs to a family of zinc finger proteins that may include additional ARF-GAPs. Gene deletion experiments of other family members showed that only GLO3 deletion resulted in impaired retrieval of dilysine-tagged proteins back to the ER. These results demonstrate that Glo3p is the main ARF-GAP specifically involved in ER retrieval.

Self-organized growth of metallic nanoparticles in a thin film under homogeneous and continuous-wave light excitation

Using a monochromatic plane wave to generate periodic arrays of metallic nanoparticles with tunable features buried in thin films is the original work we report here. We focus on the way such waveguiding metallic photonic crystals can self-emerge from thin films homogeneously loaded with metallic precursors under continuous-wave and homogeneous laser excitation. This paper fully describes the conditions leading to the formation of periodic structures and highlights the role of several parameters in the underlying physical mechanisms. The laser exposure parameters, especially, fix the geometrical and optical properties of the generated structures. Grating lines are parallel to the laser polarization and the period is directly linked to the laser wavelength. Both electron resonances of metal nanoparticles and optical resonances of guided modes interact to form the periodic patterns under homogeneous exposure. A model, based on the coupled mode theory, can be proposed to predict the spontaneous generation of such periodic nanostructures. It concludes that the guided waves exponentially enhance during illumination due to a positive feedback loop with the ordered growth of particles. This process opens up new fabrication techniques for making optical devices and may find applications in various fields such as polarization imaging, displays, security or lighting.

Multicolor photochromism of silver-containing mesoporous films of amorphous or anatase TiO2

Mesoporous TiO2 films loaded with silver nanoparticles grown photocatalytically, which are initially brown, change their color under visible laser irradiations. In this article, we compare the multicolor photochromisms of amorphous and anatase phases of TiO2. The mesoporous films are impregnated with silver salt and then exposed to a low-intensity UV laser light to grow silver nanoparticles. The Ag–TiO2 films are then exposed to visible laser beams, and the influences of several exposure parameters on the photochromic behavior are examined. Most of the previous studies have reported a poor stability of the photoinduced colors under day light or even in the dark, and few of them demonstrated the ability to get various colors on the same sample. These inconveniences limit the application field of such materials. On the other hand, except in our previous studies, only crystalline TiO2 is generally used, in its anatase or rutile phase. In this article we show that mesoporous films of amorphous and anatase phases of TiO2 respond in an efficient manner to light excitation and that multiple colors can be obtained on both kinds of films. For the first time on such Ag–TiO2 films we show that the various photoinduced colors are stable over considerable months. Visible intensity is shown to have a significant influence on the film behavior, which was not identified in previous studies. The laser-induced spectral changes are also shown to depend on the incident laser polarization. The photochromic behaviors are characterized in terms of color changes and spectral variations. The reproducibility of the photochromic process along reduction/oxidation cycles is demonstrated, and the stability of different laser-induced colors is reported on 6-month-old samples.

Acidic clusters target transmembrane proteins to the contractile vacuole in Dictyostelium cells.

The mechanisms responsible for the targeting of transmembrane integral proteins to the contractile vacuole (CV) network in Dictyostelium discoideum are unknown. Here we show that the transfer of the cytoplasmic domain of a CV-resident protein (Rh50) to a reporter transmembrane protein (CsA) is sufficient to address the chimera (CsA-Rh50) to the CV. We identified two clusters of acidic residues responsible for this targeting, and these motifs interacted with the γ-adaptin AP-1 subunit in a yeast protein-protein interaction assay. For the first time we report the existence of an indirect transport pathway from the plasma membrane to the CV via endosomes. Upon internalization, the small fraction of CsA-Rh50 present at the cell surface was first concentrated in endosomes distinct from early and late p80-positive endosomes and then slowly transported to the CV. Together our results suggest the existence of an AP-1-dependent selective transport to the contractile vacuole in Dictyostelium.

Dichroic colored luster of laser-induced silver nanoparticle gratings buried in dense inorganic films

This paper deals with the colorimetric properties of silver nanoparticle gratings buried in a dense titania film that result from a continuous wave laser-induced self-organization process. The samples exhibit shining colors in the direction of the specular reflection, which are very sensitive to polarization. We show that a large color gamut and a tunable dichroism can be reached by varying the exposure conditions. We also discuss the physical meaning of the observed variations in the dichroism. This laser process produces a single pass marking with a micrometer resolution and could be useful for developing innovative solutions in fields like active color displays, security, polarization imaging, or design.

Identification of clathrin-adaptor medium chains in Dictyostelium discoideum: differential expression during development

Clathrin-adaptor complexes (APs) are vesicle coat components that participate in cargo selectivity and transport vesicle formation. Here we cloned and characterized apm1, apm3 and apm4 cDNAs encoding AP medium chains (mu) in D. discoideum. Amino acid comparison suggested that predicted proteins were homologous to known mu1, mu3 and mu4 subunits of mammalian APs as they shared 69, 51, and 26% identity with mouse mu1A, human mu3A and human mu4, respectively. In all chains, amino acid residues predicted to interact with tyrosine based sorting signals were conserved. Southern blot analysis indicated only one copy of each gene in D. discoideum genome. Expression of apm1 and apm3 mRNAs stayed relatively constant during vegetative growth and throughout development. In contrast, apm4 was poorly expressed in amoebae but became well detectable by RT-PCR upon cell differentiation. This regulated expression of coat proteins enlightens the importance of intracellular membrane transport vesicles during development in D. discoideum and strengthens this attractive model organism for studying the function of coat complexes in vivo.

Photochromic Ag:TiO2 thin films on PET substrate

TiO2:Ag nanocomposite thin films were prepared on plastic PET substrate in two steps. First, a sol–gel route and evaporation induced self-assembly (EISA) method were used to fabricate the TiO2 matrix. The use of thermal treatment being discordant with the plastic substrate, the mesoporosity was realized by chemical extraction or infrared annealing. Silver was then introduced in the film porosity in the form of ions by soaking the films into a silver salt. A reversible photochromic behavior was successfully demonstrated after successive cycles of UV and visible laser exposures without degrading the PET substrate. This laser-induced colour changes are based on the reversible growth and dissociation of silver nanoparticles within the titania matrix.

ERGIC-53 KKAA signal mediates endoplasmic reticulum retrieval in yeast.

Studies on the ERGIC-53 KKAA signal have revealed a new mechanism for static retention of mammalian proteins in the endoplasmic reticulum (Andersson, H., Kappeler, F., Hauri, H. P. (1999): Protein targeting to endoplasmic reticulum by dilysine signals involves direct retention in addition to retrieval. J. Biol. Chem. 274,15080 - 15084). To test if this mechanism was conserved in yeast, the ERGIC-53 KKAA signal was transferred on two different yeast reporter proteins. Making use of a genetic assay, we demonstrate that this signal induces COPI-dependent ER retrieval. ER retention of KKAA-tagged proteins was impaired in yeast mutants affected in COPI subunits. Furthermore, biochemical analysis of post-ER carbohydrate modifications detected on reporter proteins indicated that KKAA-tagged proteins recycle continuously within early compartments of the secretory pathway. Therefore in yeast, the KKAA signal might only function as a classical dilysine ER retrieval signal.