Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation.

Ezrin, a membrane cytoskeleton linker, is involved in cellular functions, including epithelial cell morphogenesis and adhesion. A mutant form of ezrin, ezrin T567D, maintains the protein in an open conformation, which when expressed in Madin-Darby canine kidney cells causes extensive formation of lamellipodia and altered cell-cell contacts at low cell density. Furthermore, these cells do not form tubules when grown in a collagen type I matrix.

Perturbation of beta1-integrin function in involuting mammary gland results in premature dedifferentiation of secretory epithelial cells.

To study the mechanism of beta1-integrin function in vivo, we have generated transgenic mouse expressing a dominant negative mutant of beta1-integrin under the control of mouse mammary tumor virus (MMTV) promoter (MMTV-beta1-cyto). Mammary glands from MMTV-beta1-cyto transgenic females present significant growth defects during pregnancy and lactation and impaired differentiation of secretory epithelial cells at the onset of lactation. We report herein that perturbation of beta1-integrin function in involuting mammary gland induced precocious dedifferentiation of the secretory epithelium, as shown by the premature decrease in beta-casein and whey acidic protein mRNA levels, accompanied by inactivation of STAT5, a transcription factor essential for mammary gland development and up-regulation of nuclear factor-kappaB, a negative regulator of STAT5 signaling.

Molecular analysis of microscopic ezrin dynamics by two-photon FRAP.

Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin-membrane linker undergoes a series of conformational changes that modulate its interactions with various partners and its localization in membrane or cytosolic pools. Its mobility and exchange rates within and between these two pools were assessed by two-photon fluorescence recovery after photobleaching in epithelial cell microvilli.

Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein.

The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site.

A minimal system allowing tubulation with molecular motors pulling on giant liposomes.

The elucidation of physical and molecular mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that molecular motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin molecules have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP.

An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells.

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex.

Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy.

Fluorescence videomicroscopy and scanning force microscopy were used to follow, in real time, chromatin assembly on individual DNA molecules immersed in cell-free systems competent for physiological chromatin assembly. Within a few seconds, molecules are already compacted into a form exhibiting strong similarities to native chromatin fibers. In these extracts, the compaction rate is more than 100 times faster than expected from standard biochemical assays.

Auditory sensitivity provided by self-tuned critical oscillations of hair cells.

We introduce the concept of self-tuned criticality as a general mechanism for signal detection in sensory systems. In the case of hearing, we argue that active amplification of faint sounds is provided by a dynamical system that is maintained at the threshold of an oscillatory instability. This concept can account for the exquisite sensitivity of the auditory system and its wide dynamic range as well as its capacity to respond selectively to different frequencies.

Retrovirally mediated IFN-beta transduction of macrophages induces resistance to HIV, correlated with up-regulation of RANTES production and down-regulation of C-C chemokine receptor-5 expression.

Constitutive expression of IFN-beta by HIV target cells may be an alternative or complementary therapeutic approach for the treatment of AIDS. We show that macrophages derived from CD34+ cells from umbilical cord blood can be efficiently transduced by a retroviral vector carrying the IFN-beta coding sequence. This results in resistance to infection by a macrophage-tropic HIV type 1, as shown by the drastic reduction in the HIV DNA copy number per cell and in p24 release.

Dynamics of transient pores in stretched vesicles.

We image macroscopic transient pores in mechanically stretched giant vesicles. Holes open above a critical radius r(c1), grow up to a radius r(c2), and close. We interpret the upper limit r(c2) by a relaxation of the membrane tension as the holes expand.

Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway.

ERM (Ezrin-Radixin-Moesin) proteins function as plasma membrane-actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. We have investigated ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1. Here we show that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis.

Efficient in vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells.

Efficient transport of bile acids, a typical characteristic of hepatocytes, is partially lost in most hepatoma cell lines and in normal hepatocytes after some days in culture. We have tested whether the polarized rat hepatoma-human fibroblast hybrid WIF (hybrids between W138 and Fao cells) cells previously obtained by our group were able to perform vectorial transport of the fluorescent bile acid derivative cholylglycylamidofluorescein (CGamF) towards the bile canaliculi (BC). Four different WIF clones were analyzed.

The zinc-finger protein slug causes desmosome dissociation, an initial and necessary step for growth factor-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an essential morphogenetic process during embryonic development. It can be induced in vitro by hepatocyte growth factor/scatter factor (HGF/SF), or by FGF-1 in our NBT-II cell model for EMT. We tested for a central role in EMT of a zinc-finger protein called Slug.