scholarly journals A new ultradian rhythm in mammalian cell dry mass observed by holography

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lamya Ghenim ◽  
Cédric Allier ◽  
Patricia Obeid ◽  
Lionel Hervé ◽  
Jean-Yves Fortin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Dry Mass ◽  
Live Cells ◽  
Transformed Cells ◽  
Ultradian Rhythm ◽  
Cell Populations ◽  
Proliferating Cells ◽  
Chemical Reagents ◽  
Wide Range ◽  
Holographic Lens

AbstractWe have discovered a new 4 h ultradian rhythm that occurs during the interphase of the cell cycle in a wide range of individual mammalian cells, including both primary and transformed cells. The rhythm was detected by holographic lens-free microscopy that follows the histories of the dry mass of thousands of single live cells simultaneously, each at a resolution of five minutes. It was vital that the rhythm was observed in inherently heterogeneous cell populations, thus eliminating synchronization and labeling bias. The rhythm is independent of circadian rhythm, and is temperature-compensated. We show that the amplitude of the fundamental frequency provides a way to quantify the effects of, chemical reagents on cells, thus shedding light on its mechanism. The rhythm is suppressed by proteostasis disruptors and is detected only in proliferating cells, suggesting that it represents a massive degradation and re-synthesis of protein every 4 h in growing cells.

scholarly journals A New Ultradian Rhythm Linked to Protein Degradation and Synthesis in Mammalian Cells

2019 ◽  
Author(s):  
L. Ghenim ◽  
C. Allier ◽  
P. Obeid ◽  
L. Hervé ◽  
J-Y. Fortin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Dry Mass ◽  
Live Cells ◽  
Ultradian Rhythm ◽  
Proliferating Cells ◽  
Protein Mass ◽  
Wide Range ◽  
Periodic Dynamics ◽  
The Individual ◽  
Holographic Lens

AbstractWe describe a new ultradian rhythm that occurs during the interphase of the cell cycle in a wide range of mammalian cells, including both primary and transformed cells. The rhythm was detected by holographic lens-free microscopy that follows the individual histories of the dry mass of thousands of live cells simultaneously, each at a resolution of five minutes. Importantly, the rhythm was observed in inherently heterogeneous cell populations, thus eliminating synchronization and labeling bias. The rhythm is independent of circadian rhythm, has a period of 4 hours and is temperature-compensated. We demonstrated that the 4 hr rhythm is suppressed by proteostasis disruptors and is detected only in proliferating cells, suggesting that it represents the periodic dynamics of protein mass in growing cells.Brief teaserWe have revealed a 4 hr rhythm in cell dry mass dynamics that seems to be general in proliferating mammalian cells.

scholarly journals Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

2020 ◽  
Author(s):  
Peter Andrew Summers ◽  
Ben Lewis ◽  
Jorge Gonzalez-Garcia ◽  
Rosa Maria Porreca ◽  
Aaron H M Lim ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Dna Dynamics ◽  
Drug Candidates ◽  
Lifetime Imaging ◽  
Wide Range

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4 in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

scholarly journals Imaging and tracking mRNA in live mammalian cells via fluorogenic photoaffinity labeling

2020 ◽  
Author(s):  
Tewoderos M. Ayele ◽  
Travis Loya ◽  
Arielle N. Valdez-Sinon ◽  
Gary J. Bassell ◽  
Jennifer M. Heemstra
Keyword(s):  
Malachite Green ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Uv Light ◽  
Photoaffinity Labeling ◽  
Covalent Attachment ◽  
Live Cells ◽  
Future Application ◽  
Rna Labeling ◽  
Wide Range

ABSTRACTCellular RNA labeling using light-up aptamers that bind to and activate fluorogenic molecules has gained interest in recent years as an alternative to protein-based RNA labeling approaches. Aptamer-based systems are genetically encodable and cover the entire visible spectrum. However, the relatively weak nature of the non-covalent aptamer-fluorogen interaction limits the utility of these systems in that multiple copies of the aptamer are often required, and in most cases the aptamer must be expressed on a second scaffold such as a transfer RNA. We propose that these limitations can be averted through covalent RNA labeling, and here we describe a photoaffinity approach in which the aptamer ligand is functionalized with a photoactivatable reactive group such that irradiation with UV light results in covalent attachment to the RNA of interest. In addition to the robustness of the covalent linkage, this approach benefits from the ability to temporally control RNA labeling. To demonstrate this method, we incorporated a photoaffinity linker onto malachite green and fused the malachite green aptamer to a specific mRNA reporter of interest. We observed markedly improved sensitivity for fixed cell imaging of mRNA using this approach compared to in situ hybridization. Additionally, we demonstrate visualization of RNA dynamics in live cells using an mRNA having only a single copy of the aptamer, minimizing perturbation of the structure and localization. Our initial biological application utilizes the photoaffinity labeling approach to monitor RNA stress granule dynamics and we envision future application of this method for a wide range of investigations into the cellular localization, dynamics, and protein binding properties of cellular RNAs.

scholarly journals Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peter A. Summers ◽  
Benjamin W. Lewis ◽  
Jorge Gonzalez-Garcia ◽  
Rosa M. Porreca ◽  
Aaron H. M. Lim ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Dna Dynamics ◽  
Drug Candidates ◽  
Lifetime Imaging ◽  
Wide Range

AbstractGuanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

scholarly journals Intracellular Ionic Strength Sensing Using NanoLuc

2021 ◽  
Vol 22 (2) ◽  
pp. 677
Author(s):  
Tausif Altamash ◽  
Wesam Ahmed ◽  
Saad Rasool ◽  
Kabir H. Biswas
Keyword(s):  
Thermal Stability ◽  
Phase Separation ◽  
Drug Discovery ◽  
Ionic Strength ◽  
Cellular Signaling ◽  
Live Cells ◽  
Protein Activity ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Wide Range

Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

scholarly journals A novel human Smad4 mutation is involved in papillary thyroid carcinoma progression

2011 ◽  
Vol 19 (1) ◽  
pp. 39-55 ◽  
Author(s):  
Sonia D'Inzeo ◽  
Arianna Nicolussi ◽  
Caterina Francesca Donini ◽  
Massimo Zani ◽  
Patrizia Mancini ◽  
...  
Keyword(s):  
Papillary Thyroid Carcinoma ◽  
Thyroid Carcinoma ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Human Cancer ◽  
Papillary Thyroid ◽  
Tgfβ Signaling ◽  
Strong Reduction ◽  
Mesenchymal Transition ◽  
Wide Range

Smad proteins are the key effectors of the transforming growth factor β (TGFβ) signaling pathway in mammalian cells. Smad4 plays an important role in human physiology, and its mutations were found with high frequency in wide range of human cancer. In this study, we have functionally characterized Smad4 C324Y mutation, isolated from a nodal metastasis of papillary thyroid carcinoma. We demonstrated that the stable expression of Smad4 C324Y in FRTL-5 cells caused a significant activation of TGFβ signaling, responsible for the acquisition of transformed phenotype and invasive behavior. The coexpression of Smad4 C324Y with Smad4 wild-type determined an increase of homo-oligomerization of Smad4 with receptor-regulated Smads and a lengthening of nuclear localization. FRTL-5 clones overexpressing Smad4 C324Y showed a strong reduction of response to antiproliferative action of TGFβ1, acquired the ability to grow in anchorage-independent conditions, showed a fibroblast-like appearance and a strong reduction of the level of E-cadherin, one crucial event of the epithelial–mesenchymal transition process. The acquisition of a mesenchymal phenotype gave the characteristics of increased cellular motility and a significant reduction in adhesion to substrates such as fibronectin and laminin. Overall, our results demonstrate that the Smad4 C324Y mutation plays an important role in thyroid carcinogenesis and can be considered as a new prognostic and therapeutic target for thyroid cancer.

scholarly journals Primase p49 mRNA expression is serum stimulated but does not vary with the cell cycle.

1989 ◽  
Vol 9 (5) ◽  
pp. 1940-1945 ◽  
Author(s):  
B Y Tseng ◽  
C E Prussak ◽  
M T Almazan
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Small Subunit ◽  
Mrna Levels ◽  
Proliferating Cells ◽  
Restriction Point ◽  
Serum Stimulation ◽  
G1 Cells

Expression of the small-subunit p49 mRNA of primase, the enzyme that synthesizes oligoribonucleotides for initiation of DNA replication, was examined in mouse cells stimulated to proliferate by serum and in growing cells. The level of p49 mRNA increased approximately 10-fold after serum stimulation and preceded synthesis of DNA and histone H3 mRNA by several hours. Expression of p49 mRNA was not sensitive to inhibition by low concentrations of cycloheximide, which suggested that the increase in mRNA occurred before the restriction point control for cell cycle progression described for mammalian cells and was not under its control. p49 mRNA levels were not coupled to DNA synthesis, as observed for the replication-dependent histone genes, since hydroxyurea or aphidicolin had no effect on p49 mRNA levels when added before or during S phase. These inhibitors did have an effect, however, on the stability of p49 mRNA and increased the half-life from 3.5 h to about 20 h, which suggested an interdependence of p49 mRNA degradation and DNA synthesis. When growing cells were examined after separation by centrifugal elutriation, little difference was detected for p49 mRNA levels in different phases of the cell cycle. This was also observed when elutriated G1 cells were allowed to continue growth and then were blocked in M phase with colcemid. Only a small decrease in p49 mRNA occurred, whereas H3 mRNA rapidly decreased, when cells entered G2/M. These results indicate that the level of primase p49 mRNA is not cell cycle regulated but is present constitutively in proliferating cells.

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