scholarly journals Cholesterol depletion by MβCD enhances membrane tension, its heterogeneity and affects cellular integrity

2018 ◽  
Author(s):  
Arikta Biswas ◽  
Purba Kashyap ◽  
Sanchari Datta ◽  
Titas Sengupta ◽  
Bidisha Sinha
Keyword(s):  
Cellular Response ◽  
Osmotic Shock ◽  
Single Cells ◽  
Line Tension ◽  
Cholesterol Depletion ◽  
Mechanical Parameters ◽  
Membrane Tension ◽  
Membrane Cytoskeleton ◽  
Response To Stress ◽  
Tension Increase

AbstractCholesterol depletion in cells by MβCD remodels the plasma membrane’s mechanics and its interactions with the underlying cytoskeleton. Decoupling the two effects and studying various alterations to the membrane’s mechanical parameters is important for understanding cholesterol’s role in cellular response to stress. By mapping membrane height fluctuations in single cells, we report that MβCD treatment reduces temporal fluctuations and flattens out the membrane – but does not supress activity-driven fluctuations. We find that membrane tension increase contributes most to the altered fluctuations, among the multiple mechanical parameters computed. Maps also reveal an enhanced long-range heterogeneity within single cells, both in amplitude of fluctuations and membrane tension on cholesterol depletion. To check if this alters the tenacity of membrane to mechanical stress we use hypo-osmotic shock. We find that on MβCD treatment, cells are more prone to rupture than control cells, and this is not hindered by actomyosin perturbations. We report increased rupture sizes on cholesterol depletion and argue that, together, this indicates decreased lysis and line tension. Therefore, we show that cholesterol depletion directly affects cell membranes not only by enhancing membrane-cytoskeleton interactions, but also by increasing membrane tension while reducing lysis tension – hence making cells prone to rupture.

scholarly journals Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Griffin Chure ◽  
Heun Jin Lee ◽  
Akiko Rasmussen ◽  
Rob Phillips
Keyword(s):  
Cell Survival ◽  
Single Cell ◽  
Osmotic Shock ◽  
Single Cells ◽  
Mechanosensitive Channel ◽  
Membrane Tension ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Structural Methods

ABSTRACTRapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks,Escherichia coliand other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. InE. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in anE. colistrain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-typeE. colicells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than ∼100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity.IMPORTANCEMechanosensitive (MS) channels are transmembrane protein complexes which open and close in response to changes in membrane tension as a result of osmotic shock. Despite extensive biophysical characterization, the contribution of these channels to cell survival remains largely unknown. In this work, we used quantitative video microscopy to measure the abundance of a single species of MS channel in single cells, followed by their survival after a large osmotic shock. We observed total death of the population with fewer than ∼100 channels per cell and determined that approximately 500 to 700 channels were needed for 80% survival. The number of channels we found to confer nearly full survival is consistent with the counts of the numbers of channels in wild-type cells in several earlier studies. These results prompt further studies to dissect the contribution of other channel species to survival.

Use of Flexible Materials with a Novel Pressure Driven Equibiaxial Cell Stretching Device for Mechanical Stimulation of Single Mammalian Cells

2003 ◽  
Vol 773 ◽  
Author(s):  
James D. Kubicek ◽  
Stephanie Brelsford ◽  
Philip R. LeDuc
Keyword(s):  
Cell Fate ◽  
Mechanical Stimulation ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Single Cells ◽  
Complex Nature ◽  
Cellular Behavior ◽  
Cell Stretching ◽  
Stimulation Of ◽  
Pressure Driven

AbstractMechanical stimulation of single cells has been shown to affect cellular behavior from the molecular scale to ultimate cell fate including apoptosis and proliferation. In this, the ability to control the spatiotemporal application of force on cells through their extracellular matrix connections is critical to understand the cellular response of mechanotransduction. Here, we develop and utilize a novel pressure-driven equibiaxial cell stretching device (PECS) combined with an elastomeric material to control specifically the mechanical stimulation on single cells. Cells were cultured on silicone membranes coated with molecular matrices and then a uniform pressure was introduced to the opposite surface of the membrane to stretch single cells equibiaxially. This allowed us to apply mechanical deformation to investigate the complex nature of cell shape and structure. These results will enhance our knowledge of cellular and molecular function as well as provide insights into fields including biomechanics, tissue engineering, and drug discovery.

scholarly journals Deficit of mitogen-activated protein kinase phosphatase 1 (DUSP1) accelerates progressive hearing loss

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Adelaida M Celaya ◽  
Isabel Sánchez-Pérez ◽  
Jose M Bermúdez-Muñoz ◽  
Lourdes Rodríguez-de la Rosa ◽  
Laura Pintado-Berninches ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Cellular Response ◽  
Redox Status ◽  
Mitogen Activated Protein Kinase ◽  
Progressive Hearing Loss ◽  
Knock Out ◽  
Auditory Evoked Responses ◽  
Mitogen Activated Protein ◽  
Response To Stress ◽  
Stress Signals

Mitogen-activated protein kinases (MAPK) such as p38 and the c-Jun N-terminal kinases (JNKs) are activated during the cellular response to stress signals. Their activity is regulated by the MAPK-phosphatase 1 (DUSP1), a key component of the anti-inflammatory response. Stress kinases are well-described elements of the response to otic injury and the otoprotective potential of JNK inhibitors is being tested in clinical trials. By contrast, there are no studies exploring the role of DUSP1 in hearing and hearing loss. Here we show that Dusp1 expression is age-regulated in the mouse cochlea. Dusp1 gene knock-out caused premature progressive hearing loss, as confirmed by auditory evoked responses in Dusp1–/– mice. Hearing loss correlated with cell death in hair cells, degeneration of spiral neurons and increased macrophage infiltration. Dusp1–/– mouse cochleae showed imbalanced redox status and dysregulated expression of cytokines. These data suggest that DUSP1 is essential for cochlear homeostasis in the response to stress during ageing.

Role played by BRCA1 in regulating the cellular response to stress

2003 ◽  
Vol 31 (1) ◽  
pp. 257-262 ◽  
Author(s):  
P.M. Gilmore ◽  
J.E. Quinn ◽  
P.B. Mullan ◽  
H.N. Andrews ◽  
N. McCabe ◽  
...  
Keyword(s):  
Cellular Response ◽  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Breast Cancer Susceptibility ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Breast Cancer Susceptibility Gene ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
Response To Stress

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor gene that is mutated in the germline of women with a genetic predisposition to breast and ovarian cancer. In this review, we examine the role played by BRCA1 in mediating the cellular response to stress. We review the role played by BRCA1 in detecting and signalling the presence of DNA damage, particularly double-strand DNA breaks, and look at the evidence to support a role for BRCA1 in regulating stress response pathways such as the c-Jun N-terminal kinase/stress-activated protein kinase pathway. In addition, we examine the role played by BRCA1 in mediating both cell-cycle arrest and apoptosis following different types of cellular insult, and how this may be modulated by the presence or absence of associated proteins such as p53. Finally, we explore the possibility that many of the functions associated with BRCA1 may be based on transcriptional regulation of key downstream genes that have been implicated in the regulation of these specific cellular pathways.

scholarly journals Identification of the C3a Receptor (C3AR1) as the Target of the VGF-derived Peptide TLQP-21 in Rodent Cells

2013 ◽  
Vol 288 (38) ◽  
pp. 27434-27443 ◽  
Author(s):  
Sebastien Hannedouche ◽  
Valerie Beck ◽  
Juliet Leighton-Davies ◽  
Martin Beibel ◽  
Guglielmo Roma ◽  
...  
Keyword(s):  
G Protein ◽  
Cellular Response ◽  
G Protein Coupled Receptors ◽  
Proteolytic Processing ◽  
Rodent Models ◽  
Cellular Functions ◽  
Genome Wide ◽  
Response To Stress ◽  
G Protein Coupled ◽  
Human Receptor

TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation, and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This study describes the use of a series of studies that show G protein-coupled receptor-mediated biological activity of TLQP-21 signaling in CHO-K1 cells. Unbiased genome-wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible G protein-coupled receptors bringing about this activity. Further experiments using a series of defined receptor antagonists and siRNAs led to the identification of complement C3a receptor-1 (C3AR1) as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signaling to pertussis toxin is consistent with the known signaling pathway of C3AR1. The binding of TLQP-21 to C3AR1 not only has effects on signaling but also modulates cellular functions, as TLQP-21 was shown to have a role in directing migration of mouse RAW264.7 cells.

Role played by BRCA1 in regulating the cellular response to stress

2001 ◽  
Vol 31 (1) ◽  
pp. 257 ◽  
Author(s):  
P.M. Gilmore ◽  
J.E. Quinn ◽  
P.B. Mullan ◽  
H.N. Andrews ◽  
N. McCabe ◽  
...  
Keyword(s):  
Cellular Response ◽  
Response To Stress

scholarly journals Effects of diethyldithiocarbamate and endogenous polyamine content on cellular responses to hydrogen peroxide cytotoxicity

1989 ◽  
Vol 260 (2) ◽  
pp. 487-490 ◽  
Author(s):  
P M Harari ◽  
M E Tome ◽  
D J M Fuller ◽  
S W Carper ◽  
E W Gerner
Keyword(s):  
Heat Shock ◽  
Cellular Response ◽  
Chinese Hamster ◽  
Resting Cells ◽  
H2o2 Treatment ◽  
Polyamine Content ◽  
Response To Stress ◽  
Stress Dependent ◽  
Common Facet ◽  
Stimulation Of

In exponential-phase Chinese-hamster cells, 0.1 mM-diethyldithiocarbamate (DDC) afforded greater than 1 log survival protection to cultures treated before and during exposure to 1 mM-H2O2. Both DDC and H2O2 treatment stimulated the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, within 4 h of exposure. DDC, and to a lesser degree H2O2, also stimulated the activity of spermidine N1-acetyltransferase (SAT), the rate-limiting enzyme in polyamine catabolism. The increase in SAT activity, after exposure to DDC or another stress (heat shock), was inhibited in cells depleted of putrescine and spermidine by alpha-difluoromethylornithine (DFMO), the enzyme-activated suicide inhibitor of ODC. Pretreatment with DFMO or heat shock also induced resistance to H2O2 cytotoxicity. Since SAT activity is low in resting cells, yet stimulation of enzyme activity depends on endogenous spermidine pools, these results suggest that the expression of SAT activity occurs by a mechanism involving a stress-dependent displacement of spermidine into a new intracellular compartment. The stimulation of ODC and SAT activities does not appear to be a necessary component of the mechanism by which DDC protects cells from H2O2 cytotoxicity, although spermidine displacement may be a common facet of the cellular response to stress.

scholarly journals Expression profiling indicating low selenium-sensitive microRNA levels linked to cell cycle and cell stress response pathways in the CaCo-2 cell line

2017 ◽  
Vol 117 (9) ◽  
pp. 1212-1221 ◽  
Author(s):  
Mark J. McCann ◽  
Kunjana Rotjanapun ◽  
John E. Hesketh ◽  
Nicole C. Roy
Keyword(s):  
Cell Cycle ◽  
Stress Response ◽  
Cell Line ◽  
Cellular Response ◽  
Biological Pathways ◽  
Response To Stress ◽  
Low Selenium ◽  
Cell Stress Response ◽  
Canonical Wnt ◽  
Cellular Dysfunction

AbstractSe is an essential micronutrient for human health, and fluctuations in Se levels and the potential cellular dysfunction associated with it may increase the risk for disease. Although Se has been shown to influence several biological pathways important in health, little is known about the effect of Se on the expression of microRNA (miRNA) molecules regulating these pathways. To explore the potential role of Se-sensitive miRNA in regulating pathways linked with colon cancer, we profiled the expression of 800 miRNA in the CaCo-2 human adenocarcinoma cell line in response to a low-Se (72 h at <40 nm) environment using nCounter direct quantification. These data were then examined using a range ofin silicodatabases to identify experimentally validated miRNA–mRNA interactions and the biological pathways involved. We identified ten Se-sensitive miRNA (hsa-miR-93-5p, hsa-miR-106a-5p, hsa-miR-205-5p, hsa-miR-200c-3p, hsa-miR-99b-5p, hsa-miR-302d-3p, hsa-miR-373-3p, hsa-miR-483-3p, hsa-miR-512-5p and hsa-miR-4454), which regulate 3588 mRNA in key pathways such as the cell cycle, the cellular response to stress, and the canonical Wnt/β-catenin, p53 and ERK/MAPK signalling pathways. Our data show that the effects of low Se on biological pathways may, in part, be due to these ten Se-sensitive miRNA. Dysregulation of the cell cycle and of the stress response pathways due to low Se may influence key genes involved in carcinogenesis.

scholarly journals Autophagy: molecular machinery, regulation, and implications for renal pathophysiology

2009 ◽  
Vol 297 (2) ◽  
pp. F244-F256 ◽  
Author(s):  
Sudharsan Periyasamy-Thandavan ◽  
Man Jiang ◽  
Patricia Schoenlein ◽  
Zheng Dong
Keyword(s):  
Mammalian Cells ◽  
Cellular Response ◽  
Nutrient Deprivation ◽  
Experimental Conditions ◽  
Induce Cell Death ◽  
The Core ◽  
Molecular Machinery ◽  
Response To Stress ◽  
Membrane Bound ◽  
And Function

Autophagy is a cellular process of “self-eating.” During autophagy, a portion of cytoplasm is enveloped in double membrane-bound structures called autophagosomes, which undergo maturation and fusion with lysosomes for degradation. At the core of the molecular machinery of autophagy is a specific family of genes or proteins called Atg. Originally identified in yeast, Atg orthologs are now being discovered in mammalian cells and have been shown to play critical roles in autophagy. Traditionally, autophagy is recognized as a cellular response to nutrient deprivation or starvation whereby cells digest cytoplasmic organelles and macromolecules to recycle nutrients for self-support. However, studies during the last few years have indicated that autophagy is a general cellular response to stress. Interestingly, depending on experimental conditions, especially stress levels, autophagy can directly induce cell death or act as a mechanism of cell survival. In this review, we discuss the molecular machinery, regulation, and function of autophagy. In addition, we analyze the recent findings of autophagy in renal systems and its possible role in renal pathophysiology.

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