scholarly journals A Micropatterning Strategy to Study Nuclear Mechanotransduction in Cells

Micromachines ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 810 ◽  
Author(s):  
Markville Bautista ◽  
Anthony Fernandez ◽  
Fabien Pinaud
Keyword(s):  
Nuclear Envelope ◽  
Microcontact Printing ◽  
Skin Fibroblasts ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
Network Cell ◽  
Cell Micropatterning ◽  
Nuclear Responses ◽  
Cell Adhesion And Proliferation

Micropatterning techniques have been widely used in biology, particularly in studies involving cell adhesion and proliferation on different substrates. Cell micropatterning approaches are also increasingly employed as in vitro tools to investigate intracellular mechanotransduction processes. In this report, we examined how modulating cellular shapes on two-dimensional rectangular fibronectin micropatterns of different widths influences nuclear mechanotransduction mediated by emerin, a nuclear envelope protein implicated in Emery–Dreifuss muscular dystrophy (EDMD). Fibronectin microcontact printing was tested onto glass coverslips functionalized with three different silane reagents (hexamethyldisilazane (HMDS), (3-Aminopropyl)triethoxysilane (APTES) and (3-Glycidyloxypropyl)trimethoxysilane (GPTMS)) using a vapor-phase deposition method. We observed that HMDS provides the most reliable printing surface for cell micropatterning, notably because it forms a hydrophobic organosilane monolayer that favors the retainment of surface antifouling agents on the coverslips. We showed that, under specific mechanical cues, emerin-null human skin fibroblasts display a significantly more deformed nucleus than skin fibroblasts expressing wild type emerin, indicating that emerin plays a crucial role in nuclear adaptability to mechanical stresses. We further showed that proper nuclear responses to forces involve a significant relocation of emerin from the inner nuclear envelope towards the outer nuclear envelope and the endoplasmic reticulum membrane network. Cell micropatterning by fibronectin microcontact printing directly on HMDS-treated glass represents a simple approach to apply steady-state biophysical cues to cells and study their specific mechanobiology responses in vitro.

scholarly journals Barrier-to-Autointegration Factor Phosphorylation on Ser-4 Regulates Emerin Binding to Lamin A In Vitro and Emerin Localization In Vivo

2006 ◽  
Vol 17 (3) ◽  
pp. 1154-1163 ◽  
Author(s):  
Luiza Bengtsson ◽  
Katherine L. Wilson
Keyword(s):  
Nuclear Envelope ◽  
Lamin A ◽  
Wild Type ◽  
Proliferating Cells ◽  
Positive Role ◽  
Double Stranded Dna ◽  
Transcription Regulators ◽  
Missense Mutant

Barrier-to-autointegration factor (BAF) is a conserved 10-kDa chromatin protein essential in proliferating cells. BAF dimers bind double-stranded DNA, histone H3, histone H1.1, lamin A, and transcription regulators, plus emerin and other LEM-domain nuclear proteins. Two-dimensional gel analysis showed that endogenous human and Xenopus BAF are posttranslationally modified by phosphorylation and potentially other modifications and that they are hyperphosphorylated during mitosis. The invariant Ser-4 residue on BAF is a major site of phosphorylation during both interphase and mitosis. In HeLa cells that overexpressed the phosphomimetic BAF missense mutant S4E, but not S4A, emerin mislocalized from the nuclear envelope, suggesting Ser-4-nonphosphorylated BAF normally promotes emerin localization at the nuclear envelope. Supporting this model, wild-type BAF but not mutant S4E enhanced emerin binding to lamin A in vitro. Thus, Ser-4-unphosphorylated BAF has a positive role in localizing emerin; this role may be disease relevant because loss or mislocalization of emerin causes Emery–Dreifuss muscular dystrophy. Our findings further suggest Ser-4 phosphorylation inhibits BAF binding to emerin and lamin A, and thereby weakens emerin–lamin interactions during both mitosis and interphase.

Electron Microscopy of Fibroblasts from Myotonic Muscular Dystrophy Patients

1981 ◽  
Vol 39 ◽  
pp. 498-499
Author(s):  
S. E. Miller ◽  
G. B. Hartwig ◽  
R. A. Nielsen ◽  
A. P. Frost ◽  
A. D. Roses
Keyword(s):  
Electron Microscopy ◽  
Muscular Dystrophy ◽  
Genetic Diseases ◽  
Skin Fibroblasts ◽  
Inborn Errors ◽  
Cytoplasmic Inclusions ◽  
Cultured Skin ◽  
Myotonic Muscular Dystrophy ◽  
Glycosaminoglycan Metabolism

Many genetic diseases can be demonstrated in skin cells cultured in vitro from patients with inborn errors of metabolism. Since myotonic muscular dystrophy (MMD) affects many organs other than muscle, it seems likely that this defect also might be expressed in fibroblasts. Detection of an alteration in cultured skin fibroblasts from patients would provide a valuable tool in the study of the disease as it would present a readily accessible and controllable system for examination. Furthermore, fibroblast expression would allow diagnosis of fetal and presumptomatic cases. An unusual staining pattern of MMD cultured skin fibroblasts as seen by light microscopy, namely, an increase in alcianophilia and metachromasia, has been reported; both these techniques suggest an altered glycosaminoglycan metabolism An altered growth pattern has also been described. One reference on cultured skin fibroblasts from a different dystrophy (Duchenne Muscular Dystrophy) reports increased cytoplasmic inclusions seen by electron microscopy. Also, ultrastructural alterations have been reported in muscle and thalamus biopsies from MMD patients, but no electron microscopical data is available on MMD cultured skin fibroblasts.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals InsP7is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics

2020 ◽  
Vol 117 (32) ◽  
pp. 19245-19253 ◽  
Author(s):  
Soumyadip Sahu ◽  
Zhenzhen Wang ◽  
Xinfu Jiao ◽  
Chunfang Gu ◽  
Nikolaus Jork ◽  
...  
Keyword(s):  
Stress Responses ◽  
Messenger Rna ◽  
Control Process ◽  
Stem Cell Differentiation ◽  
Wild Type ◽  
Inositol Pyrophosphate ◽  
Body Dynamics ◽  
Processing Body ◽  
The Stability

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7(5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout ofPPIP5Ks(diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e.,PPIP5KKO), which elevates cellular 5-InsP7levels by two- to threefold (i.e., within the physiological rheostatic range). ThePPIP5KKO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals Wild type GAL4 binds cooperatively to the GAL1-10 UASG in vitro

1993 ◽  
Vol 268 (13) ◽  
pp. 9629-9635
Author(s):  
T. Kang ◽  
T. Martins ◽  
I. Sadowski
Keyword(s):  
Wild Type

scholarly journals Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 878
Author(s):  
Oskar Gustafsson ◽  
Julia Rädler ◽  
Samantha Roudi ◽  
Tõnis Lehto ◽  
Mattias Hällbrink ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Cell Penetrating Peptide ◽  
Wild Type ◽  
Efficient Manner ◽  
Editing Efficiency ◽  
Freeze Thaw ◽  
Enzyme Digest ◽  
Clinical Potential ◽  
Generation Sequencing

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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