scholarly journals An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Oliver Voigt ◽  
Nicole Knabe ◽  
Sarah Nitsche ◽  
Eileen A. Erdmann ◽  
Julia Schumacher ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
High Stress ◽  
Gene Replacement ◽  
Phenotypic Traits ◽  
Solar Panels ◽  
A Cell ◽  
Black Fungi ◽  
Underlying Mechanisms ◽  
Protein Variants ◽  
Genetic Toolkit

AbstractMicrocolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast-like growth and constitutive melanin formation. They dominate a range of hostile natural and man-made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple genes with or without resistance cassettes were developed. This state-of-the-art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi.

Halide and Proton Binding Kinetics of Yellow Fluorescent Protein Variants

Biochemistry ◽  
2013 ◽  
Vol 52 (14) ◽  
pp. 2482-2491 ◽  
Author(s):  
Harriet E. Seward ◽  
Jaswir Basran ◽  
Roanne Denton ◽  
Mark Pfuhl ◽  
Frederick W. Muskett ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Binding Kinetics ◽  
Proton Binding ◽  
Protein Variants ◽  
Kinetics Of

scholarly journals Stabilization of Polar Localization of a Chemoreceptor via Its Covalent Modifications and Its Communication with a Different Chemoreceptor

2005 ◽  
Vol 187 (22) ◽  
pp. 7647-7654 ◽  
Author(s):  
Daisuke Shiomi ◽  
Satomi Banno ◽  
Michio Homma ◽  
Ikuro Kawagishi
Keyword(s):  
Histidine Kinase ◽  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Molecular Assembly ◽  
Covalent Modification ◽  
Polar Localization ◽  
Receptor Localization ◽  
A Cell ◽  
Covalent Modifications ◽  
Green Fluorescent

ABSTRACT In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.

scholarly journals The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

2014 ◽  
Vol 14 (3) ◽  
pp. 206-215 ◽  
Author(s):  
Steven Sykes ◽  
Anthony Szempruch ◽  
Stephen Hajduk
Keyword(s):  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Krebs Cycle ◽  
Content Type ◽  
African Trypanosomes ◽  
Atp Production ◽  
Cycle Enzyme ◽  
A Cell ◽  
Direct Localization ◽  
Green Fluorescent

ABSTRACT α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei . The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei . These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei .

scholarly journals Establishment and Identification of a CiPSC Lineage Reprogrammed from FSP-tdTomato Mouse Embryonic Fibroblasts (MEFs)

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Ruiping Chen ◽  
Wenxiu Xie ◽  
Baomei Cai ◽  
Yue Qin ◽  
Chuman Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Small Molecule ◽  
Fluorescent Protein ◽  
Day Treatment ◽  
Tracking System ◽  
Embryonic Stem ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Underlying Mechanisms

Safety issues associated with transcription factors or viruses may be avoided with the use of chemically induced pluripotent stem cells (CiPSCs), thus promoting their clinical application. Previously, we had successfully developed and standardized an induction method using small-molecule compound, with simple operation, uniform induction conditions, and clear constituents. In order to verify that the CiPSCs were indeed reprogrammed from mouse embryonic fibroblasts (MEFs), and further explore the underlying mechanisms, FSP-tdTomato mice were used to construct a fluorescent protein-tracking system of MEFs, for revealing the process of CiPSC reprogramming. CiPSCs were identified by morphological analysis, mRNA, and protein expression of pluripotency genes, as well as teratoma formation experiments. Results showed that after 40-day treatment of tdTomato-MEFs with small-molecule compounds, the cells were presented with prominent nucleoli, high core-to-cytoplasmic ratio, round shape, group and mass arrangement, and high expression of pluripotency gene. These cells could differentiate into three germ layer tissues in vivo. As indicated by the above results, tdTomato-MEFs could be reprogrammed into CiPSCs, a lineage that possesses pluripotency similar to mouse embryonic stem cells (mESCs), with the use of small-molecule compounds. The establishment of CiPSC lineage, tracked by fluorescent protein, would benefit further studies exploring its underlying mechanisms. With continuous expression of fluorescent proteins during cellular differentiation, this cell lineage could be used for tracking CiPSC transplantation and differentiation into functional cells.

scholarly journals Functional monopolar spindles caused by mutation in mgr, a cell division gene of Drosophila melanogaster

1988 ◽  
Vol 89 (1) ◽  
pp. 39-47 ◽  
Author(s):  
C. Gonzalez ◽  
J. Casal ◽  
P. Ripoll
Keyword(s):  
Drosophila Melanogaster ◽  
Cell Division ◽  
Genetic Analysis ◽  
Normal Chromosome ◽  
Phenotypic Traits ◽  
Polyploid Cells ◽  
A Cell

Mutation in the gene merry-go-round (mgr) of Drosophila causes a variety of phenotypic traits in somatic and germinal tissues, such as polyploid cells, metaphasic arrest, postmeiotic cysts with 16 nuclei, and spermatids with four times the normal chromosome content. The most characteristic phenotype is the appearance of mitotic and meiotic figures where all chromosomes are arranged in a circle. Treatment with anti-mitotic drugs and the phenotype of double mutants mgr asp (asp being a mutation altering the spindle) show that these circular figures need a functional spindle for their formation. These abnormal figures are caused by monopolar spindles similar to those observed after different treatments in several organisms. All mutant traits indicate that mgr performs a function necessary for the correct behaviour of centrosomes, thus opening this organelle to genetic analysis.

Crumbs2 Is an Essential Slit Diaphragm Protein of the Renal Filtration Barrier

2021 ◽  
pp. ASN.2020040501
Author(s):  
Annika Möller-Kerutt ◽  
Juan E. Rodriguez-Gatica ◽  
Karin Wacker ◽  
Rohan Bhatia ◽  
Jan-Peter Siebrasse ◽  
...  
Keyword(s):  
Er Stress ◽  
Fluorescent Protein ◽  
Glycosylation Site ◽  
Slit Diaphragm ◽  
Glycosylation Pattern ◽  
Steroid Resistant ◽  
Steroid Resistant Nephrotic Syndrome ◽  
Er Stress Response ◽  
Filtration Barrier ◽  
Protein Variants

BackgroundCrumbs2 is expressed at embryonic stages as well as in the retina, brain, and glomerular podocytes. Recent studies identifiedCRB2mutations as a novel cause of steroid-resistant nephrotic syndrome (SRNS).MethodsTo study the function of Crb2 at the renal filtration barrier, mice lacking Crb2 exclusively in podocytes were generated. Gene expression and histologic studies as well as transmission and scanning electron microscopy were used to analyze theseCrb2podKOknockout mice and their littermate controls. Furthermore, high-resolution expansion microscopy was used to investigate Crb2 distribution in murine glomeruli. For pull-down experiments, live cell imaging, and transcriptome analyses, cell lines were applied that inducibly express fluorescent protein–tagged CRB2 wild type and mutants.ResultsCrb2podKOmice developed proteinuria directly after birth that preceded a prominent development of disordered and effaced foot processes, upregulation of renal injury and inflammatory markers, and glomerulosclerosis. Pull-down assays revealed an interaction of CRB2 with Nephrin, mediated by their extracellular domains. Expansion microscopy showed that in mice glomeruli, Crb2 and Nephrin are organized in adjacent clusters. SRNS-associated CRB2 protein variants and a mutant that lacks a putative conservedO-glycosylation site were not transported to the cell surface. Instead, mutants accumulated in the ER, showed altered glycosylation pattern, and triggered an ER stress response.ConclusionsCrb2 is an essential component of the podocyte’s slit diaphragm, interacting with Nephrin. Loss of slit diaphragm targeting and increasing ER stress are pivotal factors for onset and progression of CRB2-related SRNS.

Light as a Life Tool

2020 ◽  
pp. 58-81
Author(s):  
John Parrington
Keyword(s):  
Fluorescent Protein ◽  
Green It ◽  
Live Animal ◽  
New Science ◽  
Gene Coding ◽  
A Cell ◽  
Electron Microscopes ◽  
First Time ◽  
Protein Channels ◽  
The Brain

Visual light, and radiation of other frequencies, are highly important for scientific research. The first light microscopes made it possible for the first time to see that organisms from plants to humans are composed of cells. Electron microscopes have allowed scientists to study the structural components of cells in great detail, and even determine the shapes of individual proteins. Many lifeforms also use light to attract a mate or prey, or deter an attacker. Following the identification of the gene coding for the fluorescent protein that makes certain jellyfish glow green it has become possible to use this to genetically label proteins in a living cell, or even a live animal. This means that now the location of proteins in a cell can be determined exactly. A major recent step forward in neuroscience came with the discovery of protein channels in algae that conduct ions in response to light. By creating transgenic mice that have these proteins in their brain neurons, it is now possible to modulate the activity of these neurons by shining light into the brain though microscopic fibre optic cables. This new science of optogenetics allows neurons to be switched on or off experimentally. The optogenetic approach has been used to uncover the neural circuits involved in memory, pain and pleasure. In the future this technique might be used to treat physical pain or depression in people. Controversially, it might be also be misused, to supress memories, or even create completely false ones in people’s heads.

scholarly journals Function and Benefits of Natural Competence in Cyanobacteria: From Ecology to Targeted Manipulation

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 249
Author(s):  
Alexandra M. Schirmacher ◽  
Sayali S. Hanamghar ◽  
Julie A. Z. Zedler
Keyword(s):  
Photosynthetic Bacteria ◽  
Molecular Mechanisms ◽  
Natural Competence ◽  
Genetic Potential ◽  
Cyanobacterial Species ◽  
A Cell ◽  
Foreign Dna ◽  
Underlying Mechanisms ◽  
Globally Distributed ◽  
Shed Light

Natural competence is the ability of a cell to actively take up and incorporate foreign DNA in its own genome. This trait is widespread and ecologically significant within the prokaryotic kingdom. Here we look at natural competence in cyanobacteria, a group of globally distributed oxygenic photosynthetic bacteria. Many cyanobacterial species appear to have the genetic potential to be naturally competent, however, this ability has only been demonstrated in a few species. Reasons for this might be due to a high variety of largely uncharacterised competence inducers and a lack of understanding the ecological context of natural competence in cyanobacteria. To shed light on these questions, we describe what is known about the molecular mechanisms of natural competence in cyanobacteria and analyse how widespread this trait might be based on available genomic datasets. Potential regulators of natural competence and what benefits or drawbacks may derive from taking up foreign DNA are discussed. Overall, many unknowns about natural competence in cyanobacteria remain to be unravelled. A better understanding of underlying mechanisms and how to manipulate these, can aid the implementation of cyanobacteria as sustainable production chassis.

scholarly journals The Cell Type–Specific Functions of miR-21 in Cardiovascular Diseases

2020 ◽  
Vol 11 ◽  
Author(s):  
Beibei Dai ◽  
Feng Wang ◽  
Xiang Nie ◽  
Hengzhi Du ◽  
Yanru Zhao ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Vascular System ◽  
Cardiac Fibroblasts ◽  
Pressure Overload ◽  
Cell Type ◽  
Physiological Processes ◽  
A Cell ◽  
Cell Type Specific ◽  
Underlying Mechanisms ◽  
Endothelial Nitric Oxide

Cardiovascular diseases are one of the prime reasons for disability and death worldwide. Diseases and conditions, such as hypoxia, pressure overload, infection, and hyperglycemia, might initiate cardiac remodeling and dysfunction by inducing hypertrophy or apoptosis in cardiomyocytes and by promoting proliferation in cardiac fibroblasts. In the vascular system, injuries decrease the endothelial nitric oxide levels and affect the phenotype of vascular smooth muscle cells. Understanding the underlying mechanisms will be helpful for the development of a precise therapeutic approach. Various microRNAs are involved in mediating multiple pathological and physiological processes in the heart. A cardiac enriched microRNA, miR-21, which is essential for cardiac homeostasis, has been demonstrated to act as a cell–cell messenger with diverse functions. This review describes the cell type–specific functions of miR-21 in different cardiovascular diseases and its prospects in clinical therapy.

scholarly journals Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

2015 ◽  
Vol 113 (3) ◽  
pp. 497-502 ◽  
Author(s):  
Marie-Aude Plamont ◽  
Emmanuelle Billon-Denis ◽  
Sylvie Maurin ◽  
Carole Gauron ◽  
Frederico M. Pimenta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Activation Mechanism ◽  
Photoactive Yellow Protein ◽  
Reversible Binding ◽  
A Cell ◽  
Rapid Labeling ◽  
Green Fluorescent

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

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