scholarly journals isoTarget: A Genetic Method for Analyzing the Functional Diversity of Splicing Isoforms In Vivo

Cell Reports ◽  
2020 ◽  
Vol 33 (6) ◽  
pp. 108361
Author(s):  
Hao Liu ◽  
Sarah Pizzano ◽  
Ruonan Li ◽  
Wenquan Zhao ◽  
Macy W. Veling ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Genetic Method ◽  
Splicing Isoforms

scholarly journals isoTarget: a genetic method for analyzing the functional diversity of splicing isoforms in vivo

2020 ◽  
Author(s):  
Hao Liu ◽  
Sarah Pizzano ◽  
Ruonan Li ◽  
Wenquan Zhao ◽  
Macy W. Veling ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Biochemical Properties ◽  
Protein Isoforms ◽  
Genetic Method ◽  
Axon Development ◽  
Splicing Isoforms ◽  
Multi Level ◽  
The Difference ◽  
Endogenous Proteins

SUMMARYProtein isoforms generated by alternative splicing contribute to proteome diversity. Due to the lack of effective techniques, isoform-specific functions, expression, localization, and signaling mechanisms of endogenous proteins in vivo are unknown for most genes. Here we report a genetic method, termed isoTarget, for blocking the expression of a targeted isoform without affecting the other isoforms and for conditional tagging the targeted isoform for multi-level analyses in select cells. Applying isoTarget to two mutually exclusive isoforms of Drosophila Dscam, Dscam[TM1] and [TM2], we found that endogenous Dscam[TM1] is localized in dendrites while Dscam[TM2] is in both dendrites and axons. We demonstrate that the difference in subcellular localization between Dscam[TM1] and [TM2], rather than any difference in biochemical properties, leads to the two isoforms’ differential contributions to dendrite and axon development. Moreover, with isoTarget, we discovered that the subcellular enrichment of functional partners results in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade specifically in axons. isoTarget is an effective technique for studying how alternative splicing enhances proteome complexity.

scholarly journals Phosphorylation within Intrinsic Disordered Region Discriminates Histone Variant macroH2A1 Splicing Isoforms—macroH2A1.1 and macroH2A1.2

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 659
Author(s):  
Sebastiano Giallongo ◽  
Oriana Lo Re ◽  
Gabriela Lochmanová ◽  
Luca Parca ◽  
Francesco Petrizzelli ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Element ◽  
Histone Variants ◽  
Histone H2a ◽  
Human Hepatoma Cells ◽  
Nucleosome Core ◽  
Intrinsically Disordered ◽  
Splicing Isoforms ◽  
The Impact

Background: Gene expression in eukaryotic cells can be governed by histone variants, which replace replication-coupled histones, conferring unique chromatin properties. MacroH2A1 is a histone H2A variant containing a domain highly similar to H2A and a large non-histone (macro) domain. MacroH2A1, in turn, is present in two alternatively exon-spliced isoforms: macroH2A1.1 and macroH2A1.2, which regulate cell plasticity and proliferation in a remarkably distinct manner. The N-terminal and the C-terminal tails of H2A histones stem from the nucleosome core structure and can be target sites for several post-translational modifications (PTMs). MacroH2A1.1 and macroH2A1.2 isoforms differ only in a few amino acids and their ability to bind NAD-derived metabolites, a property allegedly conferring their different functions in vivo. Some of the modifications on the macroH2A1 variant have been identified, such as phosphorylation (T129, S138) and methylation (K18, K123, K239). However, no study to our knowledge has analyzed extensively, and in parallel, the PTM pattern of macroH2A1.1 and macroH2A1.2 in the same experimental setting, which could facilitate the understanding of their distinct biological functions in health and disease. Methods: We used a mass spectrometry-based approach to identify the sites for phosphorylation, acetylation, and methylation in green fluorescent protein (GFP)-tagged macroH2A1.1 and macroH2A1.2 expressed in human hepatoma cells. The impact of selected PTMs on macroH2A1.1 and macroH2A1.2 structure and function are demonstrated using computational analyses. Results: We identified K7 as a new acetylation site in both macroH2A1 isoforms. Quantitative comparison of histone marks between the two isoforms revealed significant differences in the levels of phosphorylated T129 and S170. Our computational analysis provided evidence that the phosphorylation status in the intrinsically disordered linker region in macroH2A1 isoforms might represent a key regulatory element contributing to their distinct biological responses. Conclusions: Taken together, our results report different PTMs on the two macroH2A1 splicing isoforms as responsible for their distinct features and distribution in the cell.

scholarly journals Functional diversity for REST (NRSF) is defined by in vivo binding affinity hierarchies at the DNA sequence level

2009 ◽  
Vol 19 (6) ◽  
pp. 994-1005 ◽  
Author(s):  
A. W. Bruce ◽  
A. J. Lopez-Contreras ◽  
P. Flicek ◽  
T. A. Down ◽  
P. Dhami ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Dna Sequence ◽  
Functional Diversity ◽  
In Vivo Binding

scholarly journals In vivo functional diversity of midbrain dopamine neurons within identified axonal projections

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Navid Farassat ◽  
Kauê Machado Costa ◽  
Strahinja Stojanovic ◽  
Stefan Albert ◽  
Lora Kovacheva ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Multiple Scales ◽  
Dopamine Neurons ◽  
Unit Recording ◽  
Axonal Projections ◽  
Intact Brain ◽  
Functional Topography ◽  
Midbrain Dopamine

Functional diversity of midbrain dopamine (DA) neurons ranges across multiple scales, from differences in intrinsic properties and connectivity to selective task engagement in behaving animals. Distinct in vitro biophysical features of DA neurons have been associated with different axonal projection targets. However, it is unknown how this translates to different firing patterns of projection-defined DA subpopulations in the intact brain. We combined retrograde tracing with single-unit recording and labelling in mouse brain to create an in vivo functional topography of the midbrain DA system. We identified differences in burst firing among DA neurons projecting to dorsolateral striatum. Bursting also differentiated DA neurons in the medial substantia nigra (SN) projecting either to dorsal or ventral striatum. We found differences in mean firing rates and pause durations among ventral tegmental area (VTA) DA neurons projecting to lateral or medial shell of nucleus accumbens. Our data establishes a high-resolution functional in vivo landscape of midbrain DA neurons.

Functional diversity among 5-substituted nicotine analogs; in vitro and in vivo investigations

2002 ◽  
Vol 435 (2-3) ◽  
pp. 171-180 ◽  
Author(s):  
Małgorzata Dukat ◽  
Imad M Damaj ◽  
Richard Young ◽  
Robert Vann ◽  
Allan C Collins ◽  
...  
Keyword(s):  
Functional Diversity ◽  
Nicotine Analogs

scholarly journals Cnu, a Novel oriC-Binding Protein of Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 6998-7008 ◽  
Author(s):  
Myung Suk Kim ◽  
Sung-Hun Bae ◽  
Sang Hoon Yun ◽  
Hee Jung Lee ◽  
Sang Chun Ji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Replication Initiation ◽  
In Vitro Assays ◽  
Wild Type ◽  
Genetic Method ◽  
Dna Replication Initiation ◽  
Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

scholarly journals Neural Stimulation in vitro and in vivo by Photoacoustic Nanotransducers

2020 ◽  
Author(s):  
Yimin Huang ◽  
Ying Jiang ◽  
Xuyi Luo ◽  
Jiayingzi Wu ◽  
Haonan Zong ◽  
...  
Keyword(s):  
Acoustic Waves ◽  
Near Infrared ◽  
Neurological Diseases ◽  
Laser Pulses ◽  
Neural Stimulation ◽  
Nanosecond Laser ◽  
Genetic Method ◽  
Precise Control

AbstractNeuromodulation is an invaluable approach for study of neural circuits and clinical treatment of neurological diseases. Here, we report semiconducting polymer nanoparticles based photoacoustic nanotransducers (PANs) for neural stimulation. Our PANs strongly absorb light in the near-infrared second window and generate localized acoustic waves. PANs can also be surface-modified to selectively bind onto neurons. PAN-mediated activation of primary neurons in vitro is achieved with ten 3-nanosecond laser pulses at 1030 nm over a 3 millisecond duration. In vivo neural modulation of mouse brain activities and motor activities is demonstrated by PANs directly injected into brain cortex. With millisecond-scale temporal resolution, sub-millimeter spatial resolution and negligible heat deposition, PAN stimulation is a new non-genetic method for precise control of neuronal activities, opening potentials in non-invasive brain modulation.

scholarly journals A Chemical-genetics and Nanoparticle Enabled Approach for in vivo Protein Kinase Analysis

2020 ◽  
Author(s):  
Fengqian Chen ◽  
Qi Liu ◽  
Terrell Hilliard ◽  
Tingzeng Wang ◽  
Hongjun Liang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Intracellular Delivery ◽  
Binding Pocket ◽  
Live Cells ◽  
Genetic Method ◽  
Chemical Genetic ◽  
Methionine Residue ◽  
Two Factors

AbstractThe human kinome contains >500 protein kinases, and regulates up to 30% of the proteome. Kinase study is currently hindered by a lack of in vivo analysis approaches due to two factors: our inability to distinguish the kinase reaction of interest from those of other kinases in live cells and the cell impermeability of the ATP analogs. Herein, we tackled this issue by combining the widely used chemical genetic method developed by Dr. Kevan Shokat and colleagues with nanoparticle-mediated intracellular delivery of the ATP analog. The critical AKT1 protein kinase, which has been successfully studied with the method, was used as our initial prototype. Briefly, enlargement of the ATP binding pocket, by mutating the gate-keeper Methionine residue to a Glycine, prompted the mutant AKT1 to preferentially use the bulky ATP analog N6-Benzyl-ATP-γ-S (A*TPγS) and, thus, differentiating AKT1-catalyzed and other phosphorylation events. The lipid/calcium/phosphate (LCP) nanoparticle was used for efficient intracellular delivery of A*TPγS, overcoming the cell impermeability issue. The mutant, but not wild-type, AKT1 used the delivered A*TPγS for autophosphorylation and phosphorylating its substrates in live cells. Thus, an in vivo protein kinase analysis method has been developed. The strategy should be widely applicable to other protein kinases.

scholarly journals Cortical synaptic diversity naturally arises with heterogeneous neural populations

2020 ◽  
Author(s):  
Jacob L. Yates ◽  
Benjamin Scholl
Keyword(s):  
Functional Diversity ◽  
Dendritic Spines ◽  
Cortical Neurons ◽  
Coding Theory ◽  
Population Coding ◽  
Functional Heterogeneity ◽  
Noisy Input ◽  
Neural Populations ◽  
Extract Information

AbstractSynaptic inputs onto single cortical neurons in vivo exhibit substantial functional diversity with respect to sensory-driven activity. However, it is unclear what this diversity reflects, appearing counter-productive in generating tuned responses to specific stimuli. We propose that functional diversity naturally arises if neurons extract information encoded from noisy input populations. Focusing on a single sensory variable, orientation, we construct a probabilistic decoder that estimates orientation from the responses of a realistic hypothetical input population of neurons. Analytically derived weights exhibit diversity when input populations consist of noisy, correlated, and heterogeneous neurons. Weight diversity was necessary to accurately decode orientation. Further, in silico weight diversity matched the functional heterogeneity of dendritic spines imaged in vivo. This suggests that synaptic diversity is expected when information is extracted from realistic input populations, highlighting the importance of studying weighting structures in population coding theory and consideration in pursuits of the cortical connectome.

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