Drop Behavior Influenced by the Correlation Length on Noisy Surfaces.

We investigate numerically the role of the correlation length in drop behavior on noisy surfaces. To this aim, a phase field tool has been used. Theoretical results are confirmed by experiments of distilled water drops sitting on stainless steel and silicon surfaces textured by laser-induced periodic self-organized structures: an increase of the noise amplitude results in an amplification of the original behavior (i.e., hydrophobic is getting more hydrophobic, hydrophilic is getting more hydrophilic). Furthermore, computer simulations in two and three spatial dimensions allow for predictions of drop behavior on noisy sloped substrates under a gravitational force, a problem of large interest in controlled motion in micro- and nanofluidics.

KDM5B regulates embryonic stem cell self-renewal and represses cryptic intragenic transcription.

Although regulation of histone methylation is believed to contribute to embryonic stem cell (ESC) self-renewal, the mechanisms remain obscure. We show here that the histone H3 trimethyl lysine 4 (H3K4me3) demethylase, KDM5B, is a downstream Nanog target and critical for ESC self-renewal. Although KDM5B is believed to function as a promoter-bound repressor, we find that it paradoxically functions as an activator of a gene network associated with self-renewal. ChIP-Seq reveals that KDM5B is predominantly targeted to intragenic regions and that it is recruited to H3K36me3 via an interaction with the chromodomain protein MRG15. Depletion of KDM5B or MRG15 increases intragenic H3K4me3, increases cryptic intragenic transcription, and inhibits transcriptional elongation of KDM5B target genes. We propose that KDM5B activates self-renewal-associated gene expression by repressing cryptic initiation and maintaining an H3K4me3 gradient important for productive transcriptional elongation.

Genesis of femtosecond-induced nanostructures on solid surfaces.

The start and evolution of the formation of laser-induced periodic surface structures (LIPSS, ripples) are investigated. The important role of irradiation dose (fluence×number of pulses) for the properties of the generated structures is demonstrated. It is shown how, with an increasing dose, the structures evolve from random surface modification to regular sub-wavelength ripples, then coalesce to broader LIPSS and finally form more complex shapes when ablation produces deep craters. First experiments are presented following this evolution in one single irradiated spot.

microRNA modulation of circadian-clock period and entrainment.

microRNAs (miRNAs) are a class of small, noncoding RNAs that regulate the stability or translation of mRNA transcripts. Although recent work has implicated miRNAs in development and in disease, the expression and function of miRNAs in the adult mammalian nervous system have not been extensively characterized. Here, we examine the role of two brain-specific miRNAs, miR-219 and miR-132, in modulating the circadian clock located in the suprachiasmatic nucleus. miR-219 is a target of the CLOCK and BMAL1 complex, exhibits robust circadian rhythms of expression, and the in vivo knockdown of miR-219 lengthens the circadian period. miR-132 is induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock-gene expression, and attenuates the entraining effects of light. Collectively, these data reveal miRNAs as clock- and light-regulated genes and provide a mechanistic examination of their roles as effectors of pacemaker activity and entrainment.

Modeling of the laser polarization as control parameter in self-organized surface pattern.

To shed light on nanopattern formation upon femtosecond laser ablation, an adopted surface erosion model is developed, based on the description for ion beam sputtering. In particular, the dependence of generated patterns on the laser polarization is taken into account. We find that an asymmetry in deposition and dissipation of incident laser energy results in a respective dependence of coefficients in a nonlinear equation of the Kuramoto-Sivashinsky type. Surface morphologies obtained by this model for different polarization of the laser beam are presented and the time evolution of the nanopattern is discussed. A comparison of these numerical results with experimental data shows an excellent agreement. Dependence of femtosecond laser induced formation on the polarization of the incident beam within an adopted surface erosion model is considered. A continuum theory of erosion by polarized laser radiation is developed. We exploit the similarity to ion-beam sputtering and extend a corresponding model for laser ablation by including laser polarization. This yields a respective dependence of coefficients in a nonlinear equation of the Kuramoto-Sivashinsky type. We present the surface morphologies obtained by this model for different polarization of the laser beam and discuss a time evolution of the nanopattern. These numerical results are in a good agreement with numerous experimental data. We show that the correlation of ripples orientation with laser polarization can be described within a model where the polarization causes the breaking of symmetry at the surface. Our results support the non-linear self-organization mechanism of pattern formation on the surface of solids.

An activity-induced microRNA controls dendritic spine formation by regulating Rac1-PAK signaling.

Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synaptic plasticity remain largely uncharacterized. We show here that the CREB- and activity-regulated microRNA, miR132, is induced during periods of active synaptogenesis. Moreover, miR132 is necessary and sufficient for hippocampal spine formation. Expression of the miR132 target, p250GAP, is inversely correlated with miR132 levels and spinogenesis. Furthermore, knockdown of p250GAP increases spine formation while introduction of a p250GAP mutant unresponsive to miR132 attenuates this activity. Inhibition of miR132 decreases both mEPSC frequency and the number of GluR1-positive spines, while knockdown of p250GAP has the opposite effect. Additionally, we show that the miR132/p250GAP circuit regulates Rac1 activity and spine formation by modulating synapse-specific Kalirin7-Rac1 signaling. These data suggest that neuronal activity regulates spine formation, in part, by increasing miR132 transcription, which in turn activates a Rac1-Pak actin remodeling pathway.

An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP.

Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synapse growth and plasticity remain largely uncharacterized. Here, we show that microRNA 132 (miR132) is an activity-dependent rapid response gene regulated by the cAMP response element-binding (CREB) protein pathway. Introduction of miR132 into hippocampal neurons enhanced dendrite morphogenesis whereas inhibition of miR132 by 2'O-methyl RNA antagonists blocked these effects. Furthermore, neuronal activity inhibited translation of p250GAP, a miR132 target, and siRNA-mediated knockdown of p250GAP mimicked miR132-induced dendrite growth. Experiments using dominant-interfering mutants suggested that Rac signaling is downstream of miR132 and p250GAP. We propose that the miR132-p250GAP pathway plays a key role in activity-dependent structural and functional plasticity.

A cAMP-response element binding protein-induced microRNA regulates neuronal morphogenesis.

MicroRNAs (miRNAs) regulate cellular fate by controlling the stability or translation of mRNA transcripts. Although the spatial and temporal patterning of miRNA expression is tightly controlled, little is known about signals that induce their expression nor mechanisms of their transcriptional regulation. Furthermore, few miRNA targets have been validated experimentally. The miRNA, miR132, was identified through a genome-wide screen as a target of the transcription factor, cAMP-response element binding protein (CREB). miR132 is enriched in neurons and, like many neuronal CREB targets, is highly induced by neurotrophins. Expression of miR132 in cortical neurons induced neurite outgrowth. Conversely, inhibition of miR132 function attenuated neuronal outgrowth. We provide evidence that miR132 regulates neuronal morphogenesis by decreasing levels of the GTPase-activating protein, p250GAP. These data reveal that a CREB-regulated miRNA regulates neuronal morphogenesis by responding to extrinsic trophic cues.

Cysteine biosynthetic enzymes are the pieces of a metabolic energy pump.

Understanding the mechanisms of free energy transfer in metabolism is fundamental to understanding how the chemical forces that sustain the molecular organization of the cell are distributed. Recent studies of molecular motors (1-3) and ATP-driven proton transport (4-6) describe how chemical potential is transferred at the molecular level. These systems catalyze energy transfer through structural change and appear to be dedicated exclusively to their coupling tasks (7, 8). Here we report the discovery of a new class of energy-transfer system. It is a biosynthetic pump composed of cysteine biosynthesis enzymes, ATP sulfurylase and O-acetylserine sulfhydrylase, each with its own catalytic function and from whose interactions emerge new function: the hydrolysis of ATP. The hydrolysis is kinetically and energetically linked to the chemistry catalyzed by ATP sulfurylase, the first enzyme in the cysteine biosynthetic pathway, in such a way that each molecule of ATP hydrolyzed, each stroke of the pump, produces 1 equivalent of that enzyme's product. These findings integrate cysteine metabolism and broaden our understanding of the ways in which higher order allostery is used to effect free energy transfer.

Mts1 regulates the assembly of nonmuscle myosin-IIA.

The formation of myosin-II filaments is fundamental to contractile and motile processes in nonmuscle cells, and elucidating the mechanisms controlling filament assembly is essential for understanding how myosin-II rapidly responds to changing conditions within the cell. Several proteins including KRP and a novel 38 kDa protein (1, 2) have been shown to modulate filament assembly through the stabilization of myosin-II assemblies. In contrast, we demonstrate that mts1, a member of the Ca(2+)-regulated S100 family of proteins, may regulate the monomeric, unassembled state in an isoform-specific manner. Biochemical analyses demonstrate that mts1 has a 9-fold higher affinity for myosin-IIA filaments than for myosin-IIB filaments. At stoichiometric levels, mts1 inhibits the assembly of myosin-IIA monomers into filaments and promotes the disassembly of myosin-IIA filaments into monomers; however, mts1 has little effect on the assembly properties of myosin-IIB. Using a solution based-assay, we have demonstrated that mts1 binds to residues 1909-1924 of the myosin-IIA heavy chain, which is near the C-terminal tip of the alpha-helical coiled-coil. The observation that mts1 binds a linear sequence of approximately 16 amino acids is consistent with other S100 family members, which bind linear sequences of 13-22 residues in their protein targets. In addition, mts1 increases the critical monomer concentration for myosin-IIA filament assembly by approximately 11-fold. Kinetic assembly assays indicate that the elongation rate and the extent of polymerization depend on the initial myosin-IIA concentration; however, mts1 had only a small affect on the half-time for assembly and predominately affected the extent of myosin IIA polymerization. Altogether, these observations are consistent with mts1 regulating myosin IIA assembly by monomer sequestration and suggest that mts1 regulates cell shape and motility through the modulation of myosin-IIA function.

Solution structure of human Mts1 (S100A4) as determined by NMR spectroscopy.

Mts1 is a member of the S100 family of Ca2+-binding proteins and is implicated in promoting tumor progression and metastasis. To better understand the structure-function relationships of this protein and to begin characterizing its Ca2+-dependent interaction with protein binding targets, the three-dimensional structure of mts1 was determined in the apo state by NMR spectroscopy. As with other S100 protein family members, mts1 is a symmetric homodimer held together by noncovalent interactions between two helices from each subunit (helices 1, 4, 1', and 4') to form an X-type four-helix bundle. Each subunit of mts1 has two EF-hand Ca2+-binding domains: a pseudo-EF-hand (or S100-hand) and a typical EF-hand that are brought into proximity by a small two-stranded antiparallel beta-sheet. The S100-hand is formed by helices 1 and 2, and is similar in conformation to other members of the S100 family. In the typical EF-hand, the position of helix 3 is similar to that of another member of the S100 protein family, calcyclin (S100A6), and less like that of other S100 family members for which three-dimensional structures are available in the calcium-free state (e.g., S100B and S100A1). The differences in the position of helix 3 in the apo state of these four S100 proteins are likely due to variations in the amino acid sequence in the C-terminus of helix 4 and in loop 2 (the hinge region) and could potentially be used to subclassify the S100 protein family.

The homodimer of prostate-specific membrane antigen is a functional target for cancer therapy.

Prostate-specific membrane antigen (PSMA) is a type 2 integral membrane glycoprotein that serves as an attractive target for cancer immunotherapy by virtue of its abundant and restricted expression on the surface of prostate carcinomas and the neovasculature of most other solid tumors. However, relatively little is known about the molecular structure of this target. Here, we report that PSMA is expressed on tumor cells as a noncovalent homodimer. A truncated PSMA protein, lacking transmembrane and cytoplasmic domains, also formed homodimers, indicating that the extracellular domain is sufficient for dimerization. PSMA dimers but not monomers displayed a native conformation and possessed high-level carboxypeptidase activity. A unique dimer-specific epitope was identified by using one of a panel of novel mAbs. When used to immunize animals, dimer but not monomer elicited antibodies that efficiently recognized PSMA-expressing tumor cells. These findings on PSMA structure and biology may have important implications for active and passive immunotherapy of prostate and other cancers.