scholarly journals IntAct: a non-disruptive internal tagging strategy to study actin isoform organization and function

2021 ◽  
Author(s):  
Maxime Christie van Zwam ◽  
Willem Bosman ◽  
Wendy van Straaten ◽  
Suzanne Weijers ◽  
Emiel Seta ◽  
...  
Keyword(s):  
Living Cells ◽  
Actin Binding ◽  
Biological Processes ◽  
Epitope Tag ◽  
Actin Isoforms ◽  
Versatile Tool ◽  
Actin Isoform ◽  
Isoform Specificity ◽  
And Function

Actin plays a central role in many biological processes such as cell division, motility and contractility. In birds and mammals, actin has six, highly conserved isoforms, four of which are primarily present in muscles and two that are ubiquitously expressed across tissues. While each isoform has non-redundant biological functions, we currently lack the tools to investigate the molecular basis for isoform specificity due to their high similarity and the limited possibilities to manipulate actin. To solve this technical challenge, we developed IntAct, an internally tagged actin system to study actin isoform organization in fixed and living cells. For this, we performed a microscopy-based screen for 11 internal actin positions and identified one residue pair that allows for non-disruptive epitope tag integration. Using knockin cell lines with tags into the ubiquitously expressed β-actin, we demonstrate that IntAct actins are properly expressed and that their incorporation into filaments is indistinguishable from wildtype. We further show that IntAct actins can be visualized in living cells by exploiting the nanobody-targeted ALFA tag and that they keep their ability to interact with the actin binding proteins profilin and cofilin. Lastly, we also introduced the tag in the ubiquitously expressed γ-actin and demonstrate that the differential localization observed for actin isoforms remains unaltered for the IntAct actins. Together, our data demonstrate that IntAct is a versatile tool to study actin isoform localization, dynamics and molecular interactions to finally enable the molecular characterization of actin isoforms in biological processes.

scholarly journals Modulating dynamics and function of nuclear actin with synthetic bicyclic peptides

2020 ◽  
Author(s):  
Nanako Machida ◽  
Daisuke Takahashi ◽  
Yuya Ueno ◽  
Yoshihiro Nakama ◽  
Raphael J Gubeli ◽  
...  
Keyword(s):  
Regulation Of Gene Expression ◽  
Living Cells ◽  
Actin Binding ◽  
Nuclear Actin ◽  
Dna Double Strand Breaks ◽  
Cytoplasmic Actin ◽  
Localization Signal ◽  
Dynamics And Function ◽  
And Function ◽  
Analytical Tools

Abstract Actin exists in monomeric globular (G-) and polymerized filamentous (F-) forms and the dynamics of its polymerization/depolymerization are tightly regulated in both the cytoplasm and the nucleus. Various essential functions of nuclear actin have been identified including regulation of gene expression and involvement in the repair of DNA double-strand breaks (DSB). Small G-actin-binding molecules affect F-actin formation and can be utilized for analysis and manipulation of actin in living cells. However, these G-actin-binding molecules are obtained by extraction from natural sources or through complex chemical synthesis procedures, and therefore, the generation of their derivatives for analytical tools is underdeveloped. In addition, their effects on nuclear actin cannot be separately evaluated from those on cytoplasmic actin. Previously, we have generated synthetic bicyclic peptides, consisting of two macrocyclic rings, which bind to G-actin but not to F-actin. Here, we describe the introduction of these bicyclic peptides into living cells. Furthermore, by conjugation to a nuclear localization signal (NLS), the bicyclic peptides accumulated in the nucleus. The NLS-bicyclic peptides repress the formation of nuclear F-actin, and impair transcriptional regulation and DSB repair. These observations highlight a potential role for NLS-linked bicyclic peptides in the manipulation of dynamics and functions of nuclear actin.

A lysosome-targeting viscosity-sensitive fluorescent probe based on a novel functionalised near-infrared xanthene-indolium dye and its application in living cells

2020 ◽  
Vol 8 (38) ◽  
pp. 8838-8844
Author(s):  
Chang Liu ◽  
Tong Zhao ◽  
Song He ◽  
Liancheng Zhao ◽  
Xianshun Zeng
Keyword(s):  
Fluorescent Probe ◽  
Significant Role ◽  
Near Infrared ◽  
Living Cells ◽  
Biological Processes ◽  
The Status ◽  
And Function

The viscosity of lysosomes plays a significant role in modulating biological processes and reflects the status and function of this kind of organelle, e.g., locations, morphologies, and components.

scholarly journals Quantitative real-time imaging of glutathione

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Xiqian Jiang ◽  
Jianwei Chen ◽  
Aleksandar Bajić ◽  
Chengwei Zhang ◽  
Xianzhou Song ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Real Time ◽  
Fluorescent Probe ◽  
High Throughput ◽  
Single Cells ◽  
Living Cells ◽  
Biological Processes ◽  
Dynamic Changes ◽  
Versatile Tool

AbstractGlutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time glutathione dynamics in living cells. Using RT, we observe enhanced antioxidant capability of activated neurons and dynamic glutathione changes during ferroptosis. RT is thus a versatile tool that can be used for both confocal microscopy and flow cytometry based high-throughput quantification of glutathione levels in single cells. We envision that this new glutathione probe will enable opportunities to study glutathione dynamics and transportation and expand our understanding of the physiological and pathological roles of glutathione in living cells.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

Parasite defense mechanisms in bees: behavior, immunity, antimicrobials, and symbionts

2019 ◽  
Vol 4 (1) ◽  
pp. 59-76 ◽  
Author(s):  
Alison E. Fowler ◽  
Rebecca E. Irwin ◽  
Lynn S. Adler
Keyword(s):  
Defense Mechanisms ◽  
Poor Quality ◽  
Future Research ◽  
Immune Priming ◽  
Social Bees ◽  
Bee Health ◽  
Landscape Scales ◽  
And Function ◽  
Solitary Species

Parasites are linked to the decline of some bee populations; thus, understanding defense mechanisms has important implications for bee health. Recent advances have improved our understanding of factors mediating bee health ranging from molecular to landscape scales, but often as disparate literatures. Here, we bring together these fields and summarize our current understanding of bee defense mechanisms including immunity, immunization, and transgenerational immune priming in social and solitary species. Additionally, the characterization of microbial diversity and function in some bee taxa has shed light on the importance of microbes for bee health, but we lack information that links microbial communities to parasite infection in most bee species. Studies are beginning to identify how bee defense mechanisms are affected by stressors such as poor-quality diets and pesticides, but further research on this topic is needed. We discuss how integrating research on host traits, microbial partners, and nutrition, as well as improving our knowledge base on wild and semi-social bees, will help inform future research, conservation efforts, and management.

Gaunilo Parodies Anselm

2014 ◽  
Vol 17 (1) ◽  
pp. 45-71
Author(s):  
Geo Siegwart
Keyword(s):  
Detailed Comparison ◽  
The Third ◽  
Common Structure ◽  
Logical Reconstruction ◽  
Points Of View ◽  
And Function

The main objective is an interpretation of the island parody, in particular a logical reconstruction of the parodying argument that stays close to the text. The parodied reasoning is identified as the proof in the second chapter of the Proslogion, more specifically, this proof as it is represented by Gaunilo in the first chapter of his Liber pro insipiente. The second task is a detailed comparison between parodied and parodying argument as well as an account of their common structure. The third objective is a tentative characterization of the nature and function of parodies of arguments. It seems that parodying does not add new pertinent points of view to the usual criticism of an argument.

Preparation and Characterization of β-glucosidase Films for Stabilization and Handling in Dry Configurations

2020 ◽  
Vol 21 (8) ◽  
pp. 741-747
Author(s):  
Liguang Zhang ◽  
Yanan Shen ◽  
Wenjing Lu ◽  
Lengqiu Guo ◽  
Min Xiang ◽  
...  
Keyword(s):  
Tensile Strength ◽  
Protein Function ◽  
Protein Stabilization ◽  
Functional Protein ◽  
Elongation At Break ◽  
Gelatin Films ◽  
The Stability ◽  
And Function ◽  
General Method

Background: Although the stability of proteins is of significance to maintain protein function for therapeutical applications, this remains a challenge. Herein, a general method of preserving protein stability and function was developed using gelatin films. Method: Enzymes immobilized onto films composed of gelatin and Ethylene Glycol (EG) were developed to study their ability to stabilize proteins. As a model functional protein, β-glucosidase was selected. The tensile properties, microstructure, and crystallization behavior of the gelatin films were assessed. Result: Our results indicated that film configurations can preserve the activity of β-glucosidase under rigorous conditions (75% relative humidity and 37°C for 47 days). In both control films and films containing 1.8 % β-glucosidase, tensile strength increased with increased EG content, whilst the elongation at break increased initially, then decreased over time. The presence of β-glucosidase had a negligible influence on tensile strength and elongation at break. Scanning electron-microscopy (SEM) revealed that with increasing EG content or decreasing enzyme concentrations, a denser microstructure was observed. Conclusion: In conclusion, the dry film is a promising candidate to maintain protein stabilization and handling. The configuration is convenient and cheap, and thus applicable to protein storage and transportation processes in the future.

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