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3947 Publications
Showing 2991-3000 of 3947 resultsRing attractors are a class of recurrent networks hypothesized to underlie the representation of heading direction. Such network structures, schematized as a ring of neurons whose connectivity depends on their heading preferences, can sustain a bump-like activity pattern whose location can be updated by continuous shifts along either turn direction. We recently reported that a population of fly neurons represents the animal's heading via bump-like activity dynamics. We combined two-photon calcium imaging in head-fixed flying flies with optogenetics to overwrite the existing population representation with an artificial one, which was then maintained by the circuit with naturalistic dynamics. A network with local excitation and global inhibition enforces this unique and persistent heading representation. Ring attractor networks have long been invoked in theoretical work; our study provides physiological evidence of their existence and functional architecture.
A number of recent studies have provided compelling demonstrations that both mice and rats can be trained to perform a variety of behavioral tasks while restrained by mechanical elements mounted to the skull. The independent development of this technique by a number of laboratories has led to diverse solutions. We found that these solutions often used expensive materials and impeded future development and modification in the absence of engineering support. In order to address these issues, here we report on the development of a flexible single hardware design for electrophysiology and imaging both in brain tissue in vitro. Our hardware facilitates the rapid conversion of a single preparation between physiology and imaging system and the conversion of a given system between preparations. In addition, our use of rapid prototyping machines ("3D printers") allows for the deployment of new designs within a day. Here, we present specifications for design and manufacturing as well as some data from our lab demonstrating the suitability of the design for physiology in behaving animals and imaging in vitro and in vivo.
Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.
Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.
Neurons decentralize protein synthesis from the cell body to support the active metabolism of remote dendritic and axonal compartments. The neuronal RNA transport apparatus, composed of cis-acting RNA regulatory elements, neuronal transport granule proteins, and motor adaptor complexes, drives the long-distance RNA trafficking required for local protein synthesis. Over the past decade, advances in human genetics, subcellular biochemistry, and high-resolution imaging have implicated each member of the apparatus in several neurodegenerative diseases, establishing failed RNA transport and associated processes as a unifying pathomechanism. In this review, we deconstruct the RNA transport apparatus, exploring each constituent's role in RNA localization and illuminating their unique contributions to neurodegeneration.
Localized protein translation is critical in many biological contexts, particularly in highly polarized cells, such as neurons, to regulate gene expression in a spatiotemporal manner. The cytoplasmic polyadenylation element-binding (CPEB) family of RNA-binding proteins has emerged as a key regulator of mRNA transport and local translation required for early embryonic development, synaptic plasticity, and long-term memory (LTM). Drosophila Orb and Orb2 are single members of the CPEB1 and CPEB2 subfamilies of the CPEB proteins, respectively. At present, the identity of the mRNA targets they regulate is not fully known, and the binding specificity of the CPEB2 subfamily is a matter of debate. Using transcriptome-wide UV cross-linking and immunoprecipitation, we define the mRNA-binding sites and targets of Drosophila CPEBs. Both Orb and Orb2 bind linear cytoplasmic polyadenylation element-like sequences in the 3' UTRs of largely overlapping target mRNAs, with Orb2 potentially having a broader specificity. Both proteins use their RNA-recognition motifs but not the Zinc-finger region for RNA binding. A subset of Orb2 targets is translationally regulated in cultured S2 cells and fly head extracts. Moreover, pan-neuronal RNAi knockdown of these targets suggests that a number of these targets are involved in LTM. Our results provide a comprehensive list of mRNA targets of the two CPEB proteins in Drosophila, thus providing insights into local protein synthesis involved in various biological processes, including LTM.
The precursor primer RNA for mammalian mitochondrial DNA leading-strand replication remains as a persistent R loop formed during transcription through the mitochondrial DNA control region. We have examined model R loops, which exist in a novel and physiologically accurate preprimer conformation, as potential substrates for mammalian RNase mitochondrial RNA processing (MRP). Mouse RNase MRP accurately cleaves an R loop containing the mouse mitochondrial DNA origin. The multiple cleavage sites on the R-loop substrate match the priming sites observed in vivo, suggesting that RNase MRP alone is capable of generating virtually all of the leading-strand replication primers.
Bacterial Rho-independent terminators (RITs) are important genomic landmarks involved in gene regulation and terminating gene expression. In this investigation we present RNIE, a probabilistic approach for predicting RITs. The method is based upon covariance models which have been known for many years to be the most accurate computational tools for predicting homology in structural non-coding RNAs. We show that RNIE has superior performance in model species from a spectrum of bacterial phyla. Further analysis of species where a low number of RITs were predicted revealed a highly conserved structural sequence motif enriched near the genic termini of the pathogenic Actinobacteria, Mycobacterium tuberculosis. This motif, together with classical RITs, account for up to 90% of all the significantly structured regions from the termini of M. tuberculosis genic elements. The software, predictions and alignments described below are available from http://github.com/ppgardne/RNIE.
Regulation of gene expression is key determinant to cell structure and function. RNA localization, where specific mRNAs are transported to subcellular regions and then translated, is highly conserved in eukaryotes ranging from yeast to extremely specialized and polarized cells such as neurons. Messenger RNA and associated proteins (mRNP) move from the site of transcription in the nucleus to their final destination in the cytoplasm both passively through diffusion and actively via directed transport. Dysfunction of RNA localization, transport and translation machinery can lead to pathology. Single-molecule live-cell imaging techniques have revealed unique features of this journey with unprecedented resolution. In this review, we highlight key recent findings that have been made using these approaches and possible implications for spatial control of gene function.
The formation of neuronal connections requires the precise guidance of developing axons toward their targets. In the Drosophila visual system, photoreceptor neurons (R cells) project from the eye into the brain. These cells are grouped into some 750 clusters comprised of eight photoreceptors or R cells each. R cells fall into three classes: R1 to R6, R7, and R8. Posterior R8 cells are the first to project axons into the brain. How these axons select a specific pathway is not known. Here, we used a microarray-based approach to identify genes expressed in R8 neurons as they extend into the brain. We found that Roundabout-3 (Robo3), an axon-guidance receptor, is expressed specifically and transiently in R8 growth cones. In wild-type animals, posterior-most R8 axons extend along a border of glial cells demarcated by the expression of Slit, the secreted ligand of Robo3. In contrast, robo3 mutant R8 axons extend across this border and fasciculate inappropriately with other axon tracts. We demonstrate that either Robo1 or Robo2 rescues the robo3 mutant phenotype when each is knocked into the endogenous robo3 locus separately, indicating that R8 does not require a function unique to the Robo3 paralog. However, persistent expression of Robo3 in R8 disrupts the layer-specific targeting of R8 growth cones. Thus, the transient cell-specific expression of Robo3 plays a crucial role in establishing neural circuits in the Drosophila visual system by selectively regulating pathway choice for posterior-most R8 growth cones.