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3920 Publications
Showing 2701-2710 of 3920 resultsAMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing long-term potentiation (LTP) to increase synaptic transmission, but how AMPAR-containing vesicles are selectively trafficked to these synapses during LTP is not known. Here we developed a strategy to label AMPAR GluA1 subunits expressed from the endogenous loci of rat hippocampal neurons such that the motion of GluA1-containing vesicles in time-lapse sequences can be characterized using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of neuronal activity.
Pleiotropic genes are genes that affect more than one trait. For example, many genes required for pigmentation in the fruit fly also affect traits such as circadian rhythms, vision, and mating behavior. Here, we present evidence that two pigmentation genes, and , which encode enzymes catalyzing reciprocal reactions in the melanin biosynthesis pathway, also affect cuticular hydrocarbon (CHC) composition in females. More specifically, we report that loss-of-function mutants have a CHC profile that is biased toward long (>25C) chain CHCs, whereas loss-of-function mutants have a CHC profile that is biased toward short (<25C) chain CHCs. Moreover, pharmacological inhibition of dopamine synthesis, a key step in the melanin synthesis pathway, reversed the changes in CHC composition seen in mutants, making the CHC profiles similar to those seen in mutants. These observations suggest that genetic variation affecting and/or activity might cause correlated changes in pigmentation and CHC composition in natural populations. We tested this possibility using the Genetic Reference Panel (DGRP) and found that CHC composition covaried with pigmentation as well as levels of and expression in newly eclosed adults in a manner consistent with the and mutant phenotypes. These data suggest that the pleiotropic effects of and might contribute to covariation of pigmentation and CHC profiles in .
The proteins that regulate developmental processes in animals have generally been well conserved during evolution. A few cases are known where protein activities have functionally evolved. These rare examples raise the issue of how highly conserved regulatory proteins with many roles evolve new functions while maintaining old functions. We have investigated this by analyzing the function of the ;QA' peptide motif of the Hox protein Ultrabithorax (Ubx), a motif that has been conserved throughout insect evolution since its establishment early in the lineage. We precisely deleted the QA motif at the endogenous locus via allelic replacement in Drosophila melanogaster. Although the QA motif was originally characterized as involved in the repression of limb formation, we have found that it is highly pleiotropic. Curiously, deleting the QA motif had strong effects in some tissues while barely affecting others, suggesting that QA function is preferentially required for a subset of Ubx target genes. QA deletion homozygotes had a normal complement of limbs, but, at reduced doses of Ubx and the abdominal-A (abd-A) Hox gene, ectopic limb primordia and adult abdominal limbs formed when the QA motif was absent. These results show that redundancy and the additive contributions of activity-regulating peptide motifs play important roles in moderating the phenotypic consequences of Hox protein evolution, and that pleiotropic peptide motifs that contribute quantitatively to several functions are subject to intense purifying selection.
Developmental genes can have complex cis-regulatory regions, with multiple enhancers scattered across stretches of DNA spanning tens or hundreds of kilobases. Early work revealed remarkable modularity of enhancers, where distinct regions of DNA, bound by combinations of transcription factors, drive gene expression in defined spatio-temporal domains. Nevertheless, a few reports have shown that enhancer function may be required in multiple developmental stages, implying that regulatory elements can be pleiotropic. In these cases, it is not clear whether the pleiotropic enhancers employ the same transcription factor binding sites to drive expression at multiple developmental stages or whether enhancers function as chromatin scaffolds, where independent sets of transcription factor binding sites act at different stages. In this work we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive gene expression in multiple tissues and developmental stages at varying levels of redundancy. We have explored how this pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, whereas for a second enhancer, these roles are largely encoded by distinct transcription factor binding sites. Our data suggest that enhancer pleiotropy might be a common feature of cis-regulatory regions of developmental genes and that this pleiotropy can be encoded through multiple genetic architectures.
A pneumatic gun for ballistic delivery of microparticles to soft targets is proposed and demonstrated. The particles are accelerated by a high-speed flow of helium in a capillary tube. Vacuum suction applied to a concentric larger diameter tube is used to divert substantially all of the flow of helium from the gun nozzle, thereby preventing the gas from hitting and damaging the target. Speed of ejection of micron-sized gold particles from the gun nozzle, and their depth of penetration into agarose gels are reported.
A fundamental goal of systems neuroscience is to understand how neural activity gives rise to natural behavior. In order to achieve this goal, we must first build comprehensive models that offer quantitative descriptions of behavior. We develop a new class of probabilistic models to tackle this challenge in the study of larval zebrafish, an important model organism for neuroscience. Larval zebrafish locomote via sequences of punctate swim bouts--brief flicks of the tail--which are naturally modeled as a marked point process. However, these sequences of swim bouts belie a set of discrete and continuous internal states, latent variables that are not captured by standard point process models. We incorporate these variables as latent marks of a point process and explore various models for their dynamics. To infer the latent variables and fit the parameters of this model, we develop an amortized variational inference algorithm that targets the collapsed posterior distribution, analytically marginalizing out the discrete latent variables. With a dataset of over 120,000 swim bouts, we show that our models reveal interpretable discrete classes of swim bouts and continuous internal states like hunger that modulate their dynamics. These models are a major step toward understanding the natural behavioral program of the larval zebrafish and, ultimately, its neural underpinnings.
Recent advances in probe design have led to enhanced resolution (currently as significant as 12 nm) in optical microscopes based on near-field imaging. We demonstrate that the polarization of emitted and detected light in such microscopes can be manipulated sensitively to generate contrast. We show that the contrast on certain patterns is consistent with a simple interpretation of the requisite boundary conditions, whereas in other cases a more complicated interaction between the probe and the sample is involved. Finally application of the technique to near-filed magneto-optic imaging is demonstrated.
Centrosomes are composed of a centriolar core surrounded by a pericentriolar material (PCM) matrix that docks microtubule-nucleating γ-tubulin complexes. During mitotic entry, the PCM matrix increases in size and nucleating capacity in a process called centrosome maturation. Polo-like kinase 1 (PLK1) is recruited to centrosomes and phosphorylates PCM matrix proteins to drive their self-assembly, which leads to PCM expansion. Here, we show that in addition to controlling PCM expansion, PLK1 independently controls the generation of binding sites for γ-tubulin complexes on the PCM matrix. Selectively preventing the generation of PLK1-dependent γ-tubulin docking sites led to spindle defects and impaired chromosome segregation without affecting PCM expansion, highlighting the importance of phospho-regulated centrosomal γ-tubulin docking sites in spindle assembly. Inhibiting both γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulated the effects of loss of centrosomal PLK1 on the ability of centrosomes to catalyze spindle assembly.
BAF and PBAF are two related mammalian chromatin remodeling complexes essential for gene expression and development. PBAF, but not BAF, is able to potentiate transcriptional activation in vitro mediated by nuclear receptors, such as RXRalpha, VDR, and PPARgamma. Here we show that the ablation of PBAF-specific subunit BAF180 in mouse embryos results in severe hypoplastic ventricle development and trophoblast placental defects, similar to those found in mice lacking RXRalpha and PPARgamma. Embryonic aggregation analyses reveal that in contrast to PPARgamma-deficient mice, the heart defects are likely a direct result of BAF180 ablation, rather than an indirect consequence of trophoblast placental defects. We identified potential target genes for BAF180 in heart development, such as S100A13 as well as retinoic acid (RA)-induced targets RARbeta2 and CRABPII. Importantly, BAF180 is recruited to the promoter of these target genes and BAF180 deficiency affects the RA response for CRABPII and RARbeta2. These studies reveal unique functions of PBAF in cardiac chamber maturation.
Members of the tetraspanin superfamily function as transmembrane scaffold proteins that mediate the assembly of membrane proteins into specific signaling complexes. Tetraspanins also interact with each other and concentrate membrane proteins into tetraspanin-enriched microdomains (TEMs). Here we report that lens-specific tetraspanin MP20 can form multiple types of higher-order assemblies and we present crystalline arrays of MP20. When isolated in the absence of divalent cations, MP20 is solubilized predominantly in tetrameric form, whereas the presence of divalent cations during solubilization promotes the association of MP20 tetramers into higher-order species. This effect only occurs when divalent cations are present during solubilization but not when divalent cations are added to solubilized tetrameric MP20, suggesting that other factors may also be involved. When purified MP20 tetramers are reconstituted with native lens lipids in the presence of magnesium, MP20 forms two-dimensional (2D) crystals. A projection map at 18 A resolution calculated from negatively stained 2D crystals showed that the building block of the crystal is an octamer consisting of two tetramers related to each other by 2-fold symmetry. In addition to 2D crystals, reconstitution of MP20 with native lipids also produced a variety of large protein-lipid complexes, and we present three-dimensional (3D) reconstructions of the four most abundant of these complexes in negative stain. The various complexes formed by MP20 most likely reflect the many ways in which tetraspanins can interact with each other to allow formation of TEMs.