scholarly journals Reducing Myosin II and ATP-Dependent Mechanical Activity Increases Order and Stability of Intracellular Organelles

2021 ◽  
Vol 22 (19) ◽  
pp. 10369
Author(s):  
Ishay Wohl ◽  
Eilon Sherman
Keyword(s):  
Myosin Ii ◽  
Mechanical Work ◽  
Mechanical Activity ◽  
Cell Physiology ◽  
Label Free ◽  
Temporal Correlations ◽  
Related Cell ◽  
Intracellular Content ◽  
Health And Disease ◽  
Intracellular Organelles

Organization of intracellular content is affected by multiple simultaneous processes, including diffusion in a viscoelastic and structured environment, intracellular mechanical work and vibrations. The combined effects of these processes on intracellular organization are complex and remain poorly understood. Here, we studied the organization and dynamics of a free Ca++ probe as a small and mobile tracer in live T cells. Ca++, highlighted by Fluo-4, is localized in intracellular organelles. Inhibiting intracellular mechanical work by myosin II through blebbistatin treatment increased cellular dis-homogeneity of Ca++-rich features in length scale < 1.1 μm. We detected a similar effect in cells imaged by label-free bright-field (BF) microscopy, in mitochondria-highlighted cells and in ATP-depleted cells. Blebbistatin treatment also reduced the dynamics of the Ca++-rich features and generated prominent negative temporal correlations in their signals. Following Guggenberger et al. and numerical simulations, we suggest that diffusion in the viscoelastic and confined medium of intracellular organelles may promote spatial dis-homogeneity and stability of their content. This may be revealed only after inhibiting intracellular mechanical work and related cell vibrations. Our described mechanisms may allow the cell to control its organization via balancing its viscoelasticity and mechanical activity, with implications to cell physiology in health and disease.

Microscopy and Cell Biology: New Methods and New Questions

2019 ◽  
Vol 70 (1) ◽  
pp. 199-218
Author(s):  
Joshua D. Morris ◽  
Christine K. Payne
Keyword(s):  
Biological Sciences ◽  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
Cell Biology ◽  
Molecular Level ◽  
Label Free ◽  
Imaging Methods ◽  
New Methods ◽  
Multiple Imaging ◽  
Health And Disease

Understanding the cellular basis of human health and disease requires the spatial resolution of microscopy and the molecular-level details provided by spectroscopy. This review highlights imaging methods at the intersection of microscopy and spectroscopy with applications in cell biology. Imaging methods are divided into three broad categories: fluorescence microscopy, label-free approaches, and imaging tools that can be applied to multiple imaging modalities. Just as these imaging methods allow researchers to address new biological questions, progress in biological sciences will drive the development of new imaging methods. We highlight four topics in cell biology that illustrate the need for new imaging tools: nanoparticle-cell interactions, intracellular redox chemistry, neuroscience, and the increasing use of spheroids and organoids. Overall, our goal is to provide a brief overview of individual imaging methods and highlight recent advances in the use of microscopy for cell biology.

scholarly journals Generation and characterization of a lysosomally targeted, genetically encoded Ca2+-sensor

2012 ◽  
Vol 449 (2) ◽  
pp. 449-457 ◽  
Author(s):  
Hannah V. McCue ◽  
Joanna D. Wardyn ◽  
Robert D. Burgoyne ◽  
Lee P. Haynes
Keyword(s):  
Second Messengers ◽  
Eukaryotic Cell ◽  
Endocytic Pathway ◽  
Cell Physiology ◽  
Simple Method ◽  
Late Endosomes ◽  
Intracellular Organelles ◽  
Intracellular Second Messengers ◽  
The Impact

Distinct spatiotemporal Ca2+ signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+ signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the ER (endoplasmic reticulum) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide ‘trigger’ release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER. In order to investigate lysosomal-specific Ca2+ signalling a simple method for measuring lysosomal Ca2+ release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology.

ADP-Ribosylation, a Multifaceted Posttranslational Modification Involved in the Control of Cell Physiology in Health and Disease

2017 ◽  
Vol 118 (3) ◽  
pp. 1092-1136 ◽  
Author(s):  
Bernhard Lüscher ◽  
Mareike Bütepage ◽  
Laura Eckei ◽  
Sarah Krieg ◽  
Patricia Verheugd ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Cell Physiology ◽  
Adp Ribosylation ◽  
Health And Disease

scholarly journals The lamellipodium is a myosin independent mechanosensor

2017 ◽  
Author(s):  
Patrick W. Oakes ◽  
Tamara C. Bidone ◽  
Yvonne Beckham ◽  
Austin V. Skeeters ◽  
Guillermina R. Ramirez-San Juan ◽  
...  
Keyword(s):  
Cell Attachment ◽  
Myosin Ii ◽  
Adhesion Formation ◽  
Leading Edge ◽  
Cell Spreading ◽  
Substrate Stiffness ◽  
Cell Physiology ◽  
Cell Periphery ◽  
Initial Adhesion ◽  
Kinetics Of

AbstractThe ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young’s modulus of ∼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimentally observations. Together these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness sensitive processes.Significance StatementCell physiology can be regulated by the mechanics of the extracellular environment. Here, we demonstrate that cell spreading is a mechanosensitive process regulated by weak forces generated at the cell periphery and independent of motor activity. We show that stiffness sensing depends on the kinetics of the initial adhesion bonds that are subjected to forces driven by protein polymerization. This work demonstrates how the binding kinetics of adhesion molecules are sensitively tuned to a range of forces that enable mechanosensation.

Analysis of the uterine flush fluid proteome of healthy mares and mares with endometritis or fibrotic endometrial degeneration

2020 ◽  
Vol 32 (6) ◽  
pp. 572 ◽  
Author(s):  
Mariana Diel de Amorim ◽  
Firdous A. Khan ◽  
Tracey S. Chenier ◽  
Elizabeth L. Scholtz ◽  
M. Anthony Hayes
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass ◽  
Label Free ◽  
Chromatography Tandem Mass Spectrometry ◽  
Uterine Health ◽  
Liquid Chromatography Tandem Mass ◽  
Specific Proteins ◽  
Health And Disease ◽  
Flush Fluid

The objective of this study was to evaluate the differences in the uterine flush fluid proteome between healthy mares and mares with endometritis or fibrotic endometrial degeneration (FED). Uterine flush fluid samples were collected from healthy mares (n=8; oestrus n=5 and dioestrus n=3) and mares with endometritis (n=23; oestrus n=14 and dioestrus n=9) or FED (n=7; oestrus n=6 and dioestrus n=1). Proteomic analysis was performed using label-free liquid chromatography–tandem mass spectrometry. Of 216 proteins identified during oestrus, 127 were common to all three groups, one protein was exclusively detected in healthy mares, 47 proteins were exclusively detected in mares with endometritis and four proteins were exclusively detected in mares with FED. Of 188 proteins identified during dioestrus, 113 proteins were common between healthy mares and mares with endometritis, eight proteins were exclusively detected in healthy mares and 67 proteins were exclusively detected in mares with endometritis. Quantitative analysis revealed a subset of proteins differing in abundance between the three groups during oestrus and between healthy mares and mares with endometritis during dioestrus. These results provide a springboard for evaluation of specific proteins as biomarkers of uterine health and disease and for investigation of their roles in the establishment and maintenance of pregnancy.

scholarly journals The mechanosensitive Piezo1 channel mediates heart mechano-chemo transduction

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fan Jiang ◽  
Kunlun Yin ◽  
Kun Wu ◽  
Mingmin Zhang ◽  
Shiqiang Wang ◽  
...  
Keyword(s):  
Mechanical Load ◽  
Heart Function ◽  
Heart Diseases ◽  
Mechanical Stretch ◽  
Autonomic Response ◽  
Mechanical Activity ◽  
Diastolic Filling ◽  
Ros Signaling ◽  
Health And Disease

AbstractThe beating heart possesses the intrinsic ability to adapt cardiac output to changes in mechanical load. The century-old Frank–Starling law and Anrep effect have documented that stretching the heart during diastolic filling increases its contractile force. However, the molecular mechanotransduction mechanism and its impact on cardiac health and disease remain elusive. Here we show that the mechanically activated Piezo1 channel converts mechanical stretch of cardiomyocytes into Ca2+ and reactive oxygen species (ROS) signaling, which critically determines the mechanical activity of the heart. Either cardiac-specific knockout or overexpression of Piezo1 in mice results in defective Ca2+ and ROS signaling and the development of cardiomyopathy, demonstrating a homeostatic role of Piezo1. Piezo1 is pathologically upregulated in both mouse and human diseased hearts via an autonomic response of cardiomyocytes. Thus, Piezo1 serves as a key cardiac mechanotransducer for initiating mechano-chemo transduction and consequently maintaining normal heart function, and might represent a novel therapeutic target for treating human heart diseases.

scholarly journals A Label-Free Assay for Aminoacylation of tRNA

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1173
Author(s):  
Howard Gamper ◽  
Ya-Ming Hou
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Cell Growth ◽  
Quantitative Measurement ◽  
Label Free ◽  
Radioactive Amino Acid ◽  
Protein Synthesis Machinery ◽  
Wide Range ◽  
Health And Disease

Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3′-32P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3′-32P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.

scholarly journals Mitochondrial dynamics in astrocytes

2014 ◽  
Vol 42 (5) ◽  
pp. 1302-1310 ◽  
Author(s):  
Terri-Leigh Stephen ◽  
Swati Gupta-Agarwal ◽  
Josef T. Kittler
Keyword(s):  
Mitochondrial Dynamics ◽  
Spatial Dynamics ◽  
Synaptic Activity ◽  
Strategic Positioning ◽  
Neuronal Signalling ◽  
Bidirectional Communication ◽  
Cellular Excitability ◽  
Bidirectional Relationship ◽  
Health And Disease ◽  
Intracellular Organelles

Astrocytes exhibit cellular excitability through variations in their intracellular calcium (Ca2+) levels in response to synaptic activity. Astrocyte Ca2+ elevations can trigger the release of neuroactive substances that can modulate synaptic transmission and plasticity, hence promoting bidirectional communication with neurons. Intracellular Ca2+ dynamics can be regulated by several proteins located in the plasma membrane, within the cytosol and by intracellular organelles such as mitochondria. Spatial dynamics and strategic positioning of mitochondria are important for matching local energy provision and Ca2+ buffering requirements to the demands of neuronal signalling. Although relatively unresolved in astrocytes, further understanding the role of mitochondria in astrocytes may reveal more about the complex bidirectional relationship between astrocytes and neurons in health and disease. In the present review, we discuss some recent insights regarding mitochondrial function, transport and turnover in astrocytes and highlight some important questions that remain to be answered.

scholarly journals Analyzing Olfactory Neuron Precursors Non-Invasively Isolated through NADH FLIM as a Potential Tool to Study Oxidative Stress in Alzheimer’s Disease

2021 ◽  
Vol 22 (12) ◽  
pp. 6311
Author(s):  
Laura Gómez-Virgilio ◽  
Alejandro Luarte ◽  
Daniela P. Ponce ◽  
Bárbara A. Bruna ◽  
María I. Behrens
Keyword(s):  
Oxidative Stress ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Physiology ◽  
Fluorescence Lifetime Imaging ◽  
Label Free ◽  
Neuronal Precursors ◽  
Highly Correlated ◽  
Effective Diagnosis ◽  
Reduced Nicotinamide Adenine Dinucleotide

Among all the proposed pathogenic mechanisms to understand the etiology of Alzheimer’s disease (AD), increased oxidative stress seems to be a robust and early disease feature where many of those hypotheses converge. However, despite the significant lines of evidence accumulated, an effective diagnosis and treatment of AD are not yet available. This limitation might be partially explained by the use of cellular and animal models that recapitulate partial aspects of the disease and do not account for the particular biology of patients. As such, cultures of patient-derived cells of peripheral origin may provide a convenient solution for this problem. Peripheral cells of neuronal lineage such as olfactory neuronal precursors (ONPs) can be easily cultured through non-invasive isolation, reproducing AD-related oxidative stress. Interestingly, the autofluorescence of key metabolic cofactors such as reduced nicotinamide adenine dinucleotide (NADH) can be highly correlated with the oxidative state and antioxidant capacity of cells in a non-destructive and label-free manner. In particular, imaging NADH through fluorescence lifetime imaging microscopy (FLIM) has greatly improved the sensitivity in detecting oxidative shifts with minimal intervention to cell physiology. Here, we discuss the translational potential of analyzing patient-derived ONPs non-invasively isolated through NADH FLIM to reveal AD-related oxidative stress. We believe this approach may potentially accelerate the discovery of effective antioxidant therapies and contribute to early diagnosis and personalized monitoring of this devastating disease.

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