scholarly journals Chemical reporters to study mammalian O-glycosylation

2021 ◽  
Author(s):  
Kathryn E. Huxley ◽  
Lianne I. Willems
Keyword(s):  
Chemical Modification ◽  
Living Cells ◽  
Biosynthetic Pathways ◽  
Dynamic Nature ◽  
Dynamic Changes ◽  
Cellular Processes ◽  
Chemical Reporter ◽  
Native Context ◽  
Normal Cellular Function ◽  
Metabolic Oligosaccharide Engineering

Glycans play essential roles in a range of cellular processes and have been shown to contribute to various pathologies. The diversity and dynamic nature of glycan structures and the complexities of glycan biosynthetic pathways make it challenging to study the roles of specific glycans in normal cellular function and disease. Chemical reporters have emerged as powerful tools to characterise glycan structures and monitor dynamic changes in glycan levels in a native context. A variety of tags can be introduced onto specific monosaccharides via the chemical modification of endogenous glycan structures or by metabolic or enzymatic incorporation of unnatural monosaccharides into cellular glycans. These chemical reporter strategies offer unique opportunities to study and manipulate glycan functions in living cells or whole organisms. In this review, we discuss recent advances in metabolic oligosaccharide engineering and chemoenzymatic glycan labelling, focusing on their application to the study of mammalian O-linked glycans. We describe current barriers to achieving glycan labelling specificity and highlight innovations that have started to pave the way to overcome these challenges.

scholarly journals Toolbox Accelerating Glycomics (TAG): Glycan Annotation from MALDI-TOF MS Spectra and Mapping Expression Variation to Biosynthetic Pathways

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1383
Author(s):  
Nobuaki Miura ◽  
Hisatoshi Hanamatsu ◽  
Ikuko Yokota ◽  
Kazue Okada ◽  
Jun-Ichi Furukawa ◽  
...  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Structural Diversity ◽  
Biosynthetic Pathways ◽  
The Novel ◽  
Cellular Processes ◽  
Manual Analysis ◽  
Npc1 Gene ◽  
Heterogeneous Structures ◽  
Free Oligosaccharides

Glycans present extraordinary structural diversity commensurate with their involvement in numerous fundamental cellular processes including growth, differentiation, and morphogenesis. Unlike linear DNA and protein sequences, glycans have heterogeneous structures that differ in composition, branching, linkage, and anomericity. These differences pose a challenge to developing useful software for glycomic analysis. To overcome this problem, we developed the novel Toolbox Accelerating Glycomics (TAG) program. TAG consists of three units: ‘TAG List’ creates a glycan list that is used for database searching in TAG Expression; ‘TAG Expression’ automatically annotates and quantifies glycan signals and draws graphs; and ‘TAG Pathway’ maps the obtained expression information to biosynthetic pathways. Herein, we discuss the concepts, outline the TAG process, and demonstrate its potential using glycomic expression profile data from Chinese hamster ovary (CHO) cells and mutants lacking a functional Npc1 gene (Npc1 knockout (KO) CHO cells). TAG not only drastically reduced the amount of time and labor needed for glycomic analysis but also detected and quantified more glycans than manual analysis. Although this study was limited to the analysis of N-glycans and free oligosaccharides, the glycomic platform will be expanded to facilitate the analysis of O-glycans and glycans of glycosphingolipids.

Protein degradation, the main hub in the regulation of cellular polyamines

2016 ◽  
Vol 473 (24) ◽  
pp. 4551-4558 ◽  
Author(s):  
Chaim Kahana
Keyword(s):  
Central Element ◽  
Living Cells ◽  
26S Proteasome ◽  
Regulatory Proteins ◽  
Cellular Functions ◽  
Ribosomal Frameshifting ◽  
Cellular Processes ◽  
Regulatory Circuit ◽  
Polyamine Uptake ◽  
Rate Limiting

Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the biosynthesis of polyamines, low-molecular-mass aliphatic polycations that are ubiquitously present in all living cells and are essential for fundamental cellular processes. Most cellular polyamines are bound, whereas the free pools, which regulate cellular functions, are subjected to tight regulation. The regulation of the free polyamine pools is manifested by modulation of their synthesis, catabolism, uptake and excretion. A central element that enables this regulation is the rapid degradation of key enzymes and regulators of these processes, particularly that of ODC. ODC degradation is part of an autoregulatory circuit that responds to the intracellular level of the free polyamines. The driving force of this regulatory circuit is a protein termed antizyme (Az). Az stimulates the degradation of ODC and inhibits polyamine uptake. Az acts as a sensor of the free intracellular polyamine pools as it is expressed via a polyamine-stimulated ribosomal frameshifting. Az binds to monomeric ODC subunits to prevent their reassociation into active homodimers and facilitates their ubiquitin-independent degradation by the 26S proteasome. In addition, through a yet unidentified mechanism, Az inhibits polyamine uptake. Interestingly, a protein, termed antizyme inhibitor (AzI) that is highly homologous with ODC, but retains no ornithine decarboxylating activity, seems to regulate cellular polyamines through its ability to negate Az. Overall, the degradation of ODC is a net result of interactions with regulatory proteins and possession of signals that mediate its ubiquitin-independent recognition by the proteasome.

Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

2010 ◽  
Author(s):  
Kyungsuk Yum ◽  
Sungsoo Na ◽  
Yang Xiang ◽  
Ning Wang ◽  
Min-Feng Yu
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Great Promise ◽  
Biological Processes ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

Dynamic Changes in the Distribution of a Satellite Homologous to Intergenic 26-18S rDNA Spacer in the Evolution of Nicotiana

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1935-1946
Author(s):  
K Y Lim ◽  
K Skalicka ◽  
B Koukalova ◽  
R A Volkov ◽  
R Matyasek ◽  
...  
Keyword(s):  
18S Rdna ◽  
Intergenic Spacer ◽  
Nucleolar Organizer ◽  
Nucleolar Organizer Regions ◽  
Dynamic Nature ◽  
Dynamic Changes ◽  
Nicotiana Tomentosiformis ◽  
Copy Numbers ◽  
Location Patterns

Abstract An ∼135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S3 generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions.

scholarly journals Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase

2009 ◽  
Vol 187 (6) ◽  
pp. 781-790 ◽  
Author(s):  
Yoko Hayashi-Takanaka ◽  
Kazuo Yamagata ◽  
Naohito Nozaki ◽  
Hiroshi Kimura
Keyword(s):  
Chromosome Segregation ◽  
Histone Modifications ◽  
Living Cells ◽  
Preimplantation Embryos ◽  
Genome Integrity ◽  
Aurora B ◽  
Dynamic Nature ◽  
H3s10 Phosphorylation ◽  
H3 Phosphorylation ◽  
Posttranslational Histone Modifications

Posttranslational histone modifications regulate both gene expression and genome integrity. Despite the dynamic nature of these modifications, appropriate real-time monitoring systems are lacking. In this study, we developed a method to visualize histone modifications in living somatic cells and preimplantation embryos by loading fluorescently labeled specific Fab antibody fragments. The technique was used to study histone H3 Ser10 (H3S10) phosphorylation, which occurs during chromosome condensation in mitosis mediated by the aurora B kinase. In aneuploid cancer cells that frequently missegregate chromosomes, H3S10 is phosphorylated just before the chromosomes condense, whereas aurora B already accumulates in nuclei during S phase. In contrast, in nontransformed cells, phosphorylated H3S10 foci appear for a few hours during interphase, and transient exposure to an aurora B–selective inhibitor during this period induces chromosome missegregation. These results suggest that, during interphase, moderate aurora B activity or H3S10 phosphorylation is required for accurate chromosome segregation. Visualizing histone modifications in living cells will facilitate future epigenetic and cell regulation studies.

scholarly journals Constructing de Novo Biosynthetic Pathways for Chemical Synthesis inside Living Cells

Biochemistry ◽  
2011 ◽  
Vol 50 (24) ◽  
pp. 5404-5418 ◽  
Author(s):  
Amy M. Weeks ◽  
Michelle C. Y. Chang
Keyword(s):  
Chemical Synthesis ◽  
De Novo ◽  
Living Cells ◽  
Biosynthetic Pathways

scholarly journals Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors

2015 ◽  
Vol 112 (9) ◽  
pp. E937-E946 ◽  
Author(s):  
Krishna K. Midde ◽  
Nicolas Aznar ◽  
Melanie B. Laederich ◽  
Gary S. Ma ◽  
Maya T. Kunkel ◽  
...  
Keyword(s):  
Growth Factor ◽  
G Proteins ◽  
Tyrosine Kinases ◽  
Living Cells ◽  
Molecular Engineering ◽  
Fluorescent Biosensors ◽  
Exchange Factor ◽  
Cellular Processes ◽  
Single Living Cells ◽  
G Protein Coupled

Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.

scholarly journals A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking

2017 ◽  
Author(s):  
Rajarshi P Ghosh ◽  
J Matthew Franklin ◽  
Will E. Draper ◽  
Quanming Shi ◽  
Jan T. Liphardt
Keyword(s):  
Single Molecule ◽  
Precision Measurement ◽  
Single Molecules ◽  
Living Cells ◽  
Background Suppression ◽  
Molecular Heterogeneity ◽  
Single Molecule Tracking ◽  
High Brightness ◽  
Cellular Processes ◽  
State Switching

AbstractCellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.

scholarly journals RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting

2020 ◽  
Author(s):  
Shuo Han ◽  
Boxuan Simen Zhao ◽  
Samuel A. Myers ◽  
Steven A. Carr ◽  
Chuan He ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
High Specificity ◽  
Living Cells ◽  
Cellular Processes ◽  
Endogenous Protein ◽  
Wide Range ◽  
Interaction Partners ◽  
Unbiased Manner ◽  
Human Telomerase

AbstractRNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. Capitalizing on the ability of the engineered peroxidase APEX2 to identify protein interaction partners via proximity-dependent biotinylation, we used two approaches to target APEX2 to specific cellular RNAs. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were able to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured endogenous protein interaction partners of hTR, including more than a dozen proteins not previously linked to hTR. We validated the unexpected interaction between hTR and the N6-methyladenosine (m6A) demethylase ALKBH5. Further investigation showed that endogenous hTR is modified by m6A, which can be erased by ALKBH5, and that ALKBH5 influences both telomerase complex assembly and activity. These results highlight the ability of MS2- and Cas13-targeted APEX2 to identify novel RNA-protein interactions in living cells.

scholarly journals A Dynamic Affective Core to Bind the Contents, Context, and Value of Conscious Experience

2021 ◽  
Author(s):  
Kenneth T. Kishida ◽  
L. Paul Sands
Keyword(s):  
Subjective Experience ◽  
Dopaminergic System ◽  
Behavioral Control ◽  
Brain Activity ◽  
Conscious Experience ◽  
Dynamic Nature ◽  
Dynamic Changes ◽  
Open Questions ◽  
Foundational Work ◽  
The Hard Problem

The private and dynamic nature of conscious subjective experience poses an empirical challenge that has led neuroscience-based theories about consciousness to note the importance of 'the hard problem' of explaining how subjective phenomenal experience can arise from neural activity but set it aside and focus on the 'easier' problems associated with information representation and behavior. This approach leaves a major gap in our understanding of the neural mechanisms underlying conscious subjective experience and its dynamic nature. However, computational methods integrated with a variety of tools for measuring human brain activity are beginning to link dynamic changes in subjective affect with reproducible neurobehavioral signals in humans. In particular, research applying computational reinforcement learning theory has shown tremendous utility in investigating human choice behavior and the role the dopaminergic system plays in dynamic behavioral control. This research is beginning to reveal an explicit connection between the dynamics of dopaminergic signals and dynamic changes in subjective affect. However, it should be obvious that the dopaminergic system alone is not sufficient to explain all of the complexities of affective dynamics. We review foundational work, highlight current problems and open questions, and propose a Dynamic Affective Core Hypothesis that integrates advances in our understanding of the representation of the content and context of conscious experiences with our nascent understanding about how these representations acquire and retain affective subjective value.

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