PERIOD (PER) proteins are central components within the mammalian circadian oscillator, and are believed to form a negative feedback complex that inhibits their own transcription at a particular circadian phase. Phosphorylation of PER proteins regulates their stability as well as their subcellular localization. In a systematic screen, we have identified 21 phosphorylated residues of mPER2 including Ser 659, which is mutated in patients suffering from familial advanced sleep phase syndrome (FASPS). When expressing FASPS-mutated mPER2 in oscillating fibroblasts, we can phenocopy the short period and advanced phase of FASPS patients' behavior. We show that phosphorylation at Ser 659 results in nuclear retention and stabilization of mPER2, whereas phosphorylation at other sites leads to mPER2 degradation. To conceptualize our findings, we use mathematical modeling and predict that differential PER phosphorylation events can result in opposite period phenotypes. Indeed, interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants (dbt S and dbt L ) in Drosophila.[ These cell-autonomous oscillations are thought to be established by feedback loops involving transcription of clock genes and their subsequent autoregulatory transcriptional repression. In mammals, the transcription factor heterodimer CLOCK-BMAL1 activates the expression of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes via E-box enhancer elements in their promoters. PER and CRY proteins are believed to form complexes that translocate in the nucleus to inhibit their own transcription by directly interacting with the CLOCK-BMAL1 complex.Critical to the properties of this oscillator is the delay between the production of PER and CRY proteins and their autorepression. Post-translational events such as complex formation among clock proteins, nuclear import and export, regulated degradation, modulation of transcriptional activity, and chromatin modification have all been implicated in the generation of this delay (for a review, see Harms et al. 2004). In many cases, phosphorylation of clock proteins is the key step that both initiates these events and regulates their correct timing. In cyanobacteria, even the core of the circadian oscillator seems to be based on rhythmic phosphorylation and dephosphorylation of clock proteins rather than on a transcriptional-translational feedback loop (Nakajima et al.

Astrocytes play a key role in the pathogenesis of ammonia-induced neurotoxicity and hepatic encephalopathy. As shown here, ammonia induces protein tyrosine nitration in cultured rat astrocytes, which is sensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. A similar pattern of nitrated proteins is produced by NMDA. Ammonia-induced tyrosine nitration depends on a rise in [Ca2+]i, IkB degradation, and NO synthase (iNOS) induction, which are prevented by MK-801 and the intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Moreover, the increase in tyrosine nitration is blunted by L-NMMA, 1400W, uric acid, Cu, Zn-superoxide dismutase/catalase treatment, and methionine-sulfoximine, which indicate the involvement of reactive nitrogen intermediates and intracellular glutamine accumulation. Such reactive nitrogen intermediates additionally mediate ammonia-induced phosphorylation of the MAP-kinases Erk-1/Erk-2 and p38MAPK. Among the proteins, which are tyrosine -nitrated by ammonia, glyceraldehyde-3-phosphate dehydrogenase, the peripheral-type benzodiazepine receptor, Erk-1, and glutamine synthetase are identified. Ammonia-induced nitration of glutamine synthetase is associated with a loss of enzymatic activity. Astroglial protein tyrosine nitration is found in brains from rats after acute ammonia-intoxication or after portacaval anastomosis, indicating the in vivo relevance of the present findings. The production of reactive nitrogen intermediates and protein tyrosine nitration may alter astrocyte function and contribute to ammonia neurotoxicity.

Simultaneous tracking of many thousands of individual particles in live cells is possible now with the advent of high-density superresolution imaging methods. We present an approach to extract local biophysical properties of cell-particle interaction from such newly acquired large collection of data. Because classical methods do not keep the spatial localization of individual trajectories, it is not possible to access localized biophysical parameters. In contrast, by combining the high-density superresolution imaging data with the present analysis, we determine the local properties of protein dynamics. We specifically focus on AMPA receptor (AMPAR) trafficking and estimate the strength of their molecular interaction at the subdiffraction level in hippocampal dendrites. These interactions correspond to attracting potential wells of large size, showing that the high density of AMPARs is generated by physical interactions with an ensemble of cooperative membrane surface binding sites, rather than molecular crowding or aggregation, which is the case for the membrane viral glycoprotein VSVG. We further show that AMPARs can either be pushed in or out of dendritic spines. Finally, we characterize the recurrent step of influenza trajectories. To conclude, the present analysis allows the identification of the molecular organization responsible for the heterogeneities of random trajectories in cells.stochastic analysis of trajectories | dendritic spines and synapses | single particle tracking | confined diffusion R egulation of cellular physiological processes such as synaptic transmission, signal transduction relies on molecular interactions (binding and unbinding) at specific places and involves trafficking in confined local microdomains. The efficiency of these regulations crucially depends on the underlying molecular spatial organization, the study of which remains a daunting hurdle in cellular biology. Interestingly, superresolution light optical microscopy techniques for in vivo data (1-3) have allowed monitoring a large number of molecular trajectories at the single molecule level and at nanometer resolution, that can potentially reveal unique cellular organizational features. In the recent years, various techniques based on empirical characterization have emerged to track receptors (4), and estimating the mean square displacement (MSD) along isolated trajectories allowed to differentiate between free and confined diffusion (5, 6). In addition, although a large effort was dedicated to developing single molecule tracking algorithms (5, 7, 8), a general method for the analysis of the massive collection of data and for the extraction of quantitative local information is still lacking.In this article, we derive from the classical stochastic description at a molecular level, a method to extract biophysical features from high throughput superresolution data, associated with AMPA receptor (AMPAR) trafficking on neuronal cells. Indeed, neurons are organized in local microdomains characterized by morphological and functiona...

BCL-2 homology 3 (BH3)-only proteins of the BCL-2 family such as tBID and BIM EL assist BAX-type proteins to breach the permeability barrier of the outer mitochondrial membrane, thereby allowing cytoplasmic release of cytochrome c and other active inducers of cell death normally confined to the mitochondrial intermembrane space. However, the exact mechanism by which tBID and BIM EL aid BAX and its close homologues in this mitochondrial protein release remains enigmatic. Here, using pure lipid vesicles, we provide evidence that tBID acts in concert with BAX to 1) form large membrane openings through both BH3-dependent and BH3-independent mechanisms, 2) cause lipid transbilayer movement concomitant with membrane permeabilization, and 3) disrupt the lipid bilayer structure of the membrane by promoting positive monolayer curvature stress. None of these effects were observed with BAX when BIM EL was substituted for tBID. Based on these data, we propose a novel model in which tBID assists BAX not only via protein-protein but also via protein-lipid interactions to form lipidic pore-type nonbilayer structures in the outer mitochondrial membrane through which intermembrane prodeath molecules exit mitochondria during apoptosis.Mitochondria usually play a crucial role in the cellular commitment to apoptosis through the release of a variety of prodeath molecules from the intermembrane space into the cytosol (1). This process is tightly controlled by BCL-2 family proteins, which exert their function primarily, although not exclusively, at the level of the OMM 1 (2-4). Members of the BCL-2 family possess up to four conserved regions called BCL-2 homology (BH) domains and can be either proapoptotic or antiapoptotic. Based on these criteria, BCL-2 family members can be divided into three subgroups. Members of the first subgroup, exemplified by BCL-2, contain four BH domains and act predominantly as death inhibitors. Members of the second subgroup, exemplified by BAX, contain BH1-BH3 domains and promote apoptosis in most cellular contexts. Finally members of the third subgroup share only the BH3 domain (BH3-only proteins) and appear to function invariably as death agonists. Two of the most highly studied and important BH3-only proteins are BID and BIM.BID and BIM must cooperate with multidomain proapoptotic members to kill cells (5-7). However, it is unclear exactly how BID and BIM function in concert with BAX-type proteins to induce the release of mitochondrial intermembrane apoptogenic factors. One popular model holds that BID and BIM share a common mode of action via BH3-mediated binding to BAX-type proteins at the OMM (8). This physical interaction is believed to trigger a conformational change of multidomain proapoptotic members, resulting in their intramembraneous oligomerization and OMM permeabilization. Other not necessarily mutually exclusive mechanisms of action proposed for BID and BIM include (i) binding to and neutralization or reversal of prosurvival BCL-2-type family member function (6, 7, 9, 10), (ii) modulation ...

Cell penetrating peptides (CPPs) are useful tools to deliver low-molecular-weight cargoes into cells; however, their mode of uptake is still controversial. The most efficient CPPs belong to the group of arginine-rich peptides, but a systematic assessment of their potential toxicity is lacking. In this study we combined data on the membrane translocation abilities of oligo-arginines in living cells as a function of their chain length, concentration, stability and toxicity. Using confocal microscopy analysis of living cells we evaluated the transduction frequency of the L-isoforms of oligo-arginines and lysines and then monitored their associated toxicity by concomitant addition of propidium iodide. Whereas lysines showed virtually no transduction, the transduction ability of arginines increased with the number of consecutive residues and the peptide concentration, with L-R9 and L-R10 performing overall best. We further compared the L- and D-R9 isomers and found that the D-isoform always showed a higher transduction as compared to the L-counterpart in all cell types. Notably, the transduction difference between D- and L-forms was highly variable between cell types, emphasizing the need for protease-resistant peptides as vectors for drug delivery. Real-time kinetic analysis of the D- and L-isomers applied simultaneously to the cells revealed a much faster transduction for the D-variant. The latter underlies the fact that the isomers do not mix, and penetration of one peptide does not perturb the membrane in a way that gives access to the other peptide. Finally, we performed short- and long-term cell viability and cell cycle progression analyses with the protease-resistant D-R9. Altogether, our results identified concentration windows with low toxicity and high transduction efficiency, resulting in fully bioavailable intracellular peptides.

An efficient synthesis of sialic-acid-terminated glycerol dendron to chemically functionalize 2 nm and 14 nm gold nanoparticles (AuNPs) is described. These nanoparticles are highly stable and show high activity towards the inhibition of influenza virus infection. As the binding of the viral fusion protein hemagglutinin to the host cell surface is mediated by sialic acid receptors, a multivalent interaction with sialic-acid-functionalized AuNPs is expected to competitively inhibit viral infection. Electron microscopy techniques and biochemical analysis show a high binding affinity of the 14 nm AuNPs to hemagglutinin on the virus surface and, less efficiently, to isolated hemagglutinin. The functionalized AuNPs are nontoxic to the cells under the conditions studied. This approach allows a new type of molecular-imaging activity-correlation and is of particular relevance for further application in alternative antiviral therapy.

Cholesterol analogs are often used to investigate lipid trafficking and membrane organization of native cholesterol. Here, the potential of various spin (doxyl moiety) and fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group) labeled cholesterol analogs as well as of fluorescent cholestatrienol and the naturally occurring dehydroergosterol to mimic the unique properties of native cholesterol in lipid membranes was studied in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes by electron paramagnetic resonance, nuclear magnetic resonance, and fluorescence spectroscopy. As cholesterol, all analogs undergo fluctuating motions of large amplitude parallel to the bilayer normal. Native cholesterol keeps a strict orientation in the membrane with the long axis parallel to the bilayer normal. Depending on the chemical modification or the position of the label, cholesterol analogs may adopt an "up-sidedown" orientation in the membrane or may even fluctuate between "upright" and up-side-down orientation by rotational motions about the short axis not typical for native cholesterol. Those analogs are not able to induce a comparable condensation of phospholipid membranes as known for native cholesterol revealed by 2 H nuclear magnetic resonance. However, cholesterol-induced lipid condensation is one of the key properties of native cholesterol, and, therefore, a well suited parameter to assess the potential of steroid analogs to mimic cholesterol. The study points to extreme caution when studying cholesterol behavior by the respective analogs. Among seven analogs investigated, only a spin-labeled cholesterol with the doxyl group at the end of the acyl chain and the fluorophore cholestatrienol mimic cholesterol satisfactorily. Dehydroergosterol has a similar upright orientation as cholesterol and could be used at low concentration (about 1 mol %), at which its lower potential to enhance lipid packing density does not perturb membrane organization.Cholesterol constitutes a major component of mammalian cellular membranes. Its correct distribution among intracellular membranes and the plasma membrane is essential for the homeostasis of mammalian cells. Thus, intracellular trafficking plays a major role in the correct disposition of internalized cholesterol and in the regulation of cholesterol efflux (for a recent review, see Ref.

Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4°C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis.