scholarly journals An Easy-to-Fabricate Cell Stretching System Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelial Homeostasis

2020 ◽  
Author(s):  
Kevin C. Hart ◽  
Joo Yong Sim ◽  
Matthew A. Hopcroft ◽  
Daniel J. Cohen ◽  
Jiongyi Tan ◽  
...  
Keyword(s):  
Low Cost ◽  
Time Lapse ◽  
Adherent Cell ◽  
Dependent Manner ◽  
Cell Dynamics ◽  
Density Dependent ◽  
Cell Monolayers ◽  
Epithelial Homeostasis ◽  
Tissue Biology ◽  
A Cell

AbstractMechanical forces regulate many facets of tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. In this report, we show how to fabricate a simple, low-cost uniaxial stretching device with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, and a custom MATLAB code to quantify individual cell strains. Finally, as an application note using this device, we show that uniaxial stretch slows down cellular movements in an epithelial monolayer in a cell density-dependent manner that involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of tissue biology.

scholarly journals An Easy-to-Fabricate Cell Stretcher Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelia

2021 ◽  
Author(s):  
Kevin C. Hart ◽  
Joo Yong Sim ◽  
Matthew A. Hopcroft ◽  
Daniel J. Cohen ◽  
Jiongyi Tan ◽  
...  
Keyword(s):  
Low Cost ◽  
Cell Movement ◽  
Time Lapse ◽  
Adherent Cell ◽  
Dependent Manner ◽  
Cell Dynamics ◽  
Density Dependent ◽  
Cell Movements ◽  
Tissue Biology ◽  
Mechanical Regulation

Abstract Introduction Mechanical forces regulate many facets of cell and tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. Methods We show how to fabricate a simple, low-cost, uniaxial stretching device, with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, as well as a custom MATLAB code to quantify individual cell strains. Results As an application note using this device, we show that uniaxial stretch slows down cellular movements in a mammalian epithelial monolayer in a cell density-dependent manner. We demonstrate that the effect on cell movement involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). Conclusions This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of cell and tissue biology. We used this device to demonstrate the mechanical regulation of collective cell movements in epithelia.

scholarly journals Inhibition of transferrin receptor 1 transcription by a cell density response element

2005 ◽  
Vol 392 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Jian Wang ◽  
Guohua Chen ◽  
Kostas Pantopoulos
Keyword(s):  
Cell Density ◽  
Transferrin Receptor ◽  
Human Lung Cancer ◽  
Dependent Manner ◽  
Response Element ◽  
Iron Regulatory Proteins ◽  
Density Dependent ◽  
Transferrin Receptor 1 ◽  
Density Response ◽  
A Cell

TfR1 (transferrin receptor 1) mediates the uptake of transferrin-bound iron and thereby plays a critical role in cellular iron metabolism. Its expression is coupled to cell proliferation/differentiation and controlled in response to iron levels and other signals by transcriptional and post-transcriptional mechanisms. It is well established that TfR1 levels decline when cultured cells reach a high density and in the present study we have investigated the underlying mechanisms. Consistent with previous findings, we demonstrate that TfR1 expression is attenuated in a cell-density-dependent manner in human lung cancer H1299 cells and in murine B6 fibroblasts as the result of a marked decrease in mRNA content. This response is not associated with alterations in the RNA-binding activity of iron regulatory proteins that are indicative of a transcriptional mechanism. Reporter assays reveal that the human TfR1 promoters contains sequences mediating cell-density-dependent transcriptional inhibition. Mapping of the human and mouse TfR1 promoters identified a conserved hexa-nucleotide 5′-GAGGGC-3′ motif with notable sequence similarity to a previously described element within the IGF-2 (insulin-like growth factor-2) promoter. We show that this motif is necessary for the formation of specific complexes with nuclear extracts and for cell-density-dependent regulation in reporter gene assays. Thus the TfR1 promoter contains a functional ‘cell density response element’ (CDRE).

scholarly journals RalA interacts with ZONAB in a cell density-dependent manner and regulates its transcriptional activity

2004 ◽  
Vol 24 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Paul Frankel ◽  
Ami Aronheim ◽  
Emma Kavanagh ◽  
Maria S Balda ◽  
Karl Matter ◽  
...  
Keyword(s):  
Cell Density ◽  
Transcriptional Activity ◽  
Dependent Manner ◽  
Density Dependent ◽  
A Cell

scholarly journals Cytotoxicity of Brucella smooth strains for macrophages is mediated by increased secretion of the type IV secretion system

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3392-3402 ◽  
Author(s):  
Zhijun Zhong ◽  
Yufei Wang ◽  
Feng Qiao ◽  
Zhoujia Wang ◽  
Xinying Du ◽  
...  
Keyword(s):  
Cell Density ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Dependent Manner ◽  
Density Dependent ◽  
Type Iv ◽  
A Cell

Some Brucella rough mutants cause cytotoxicity that resembles oncosis and necrosis in macrophages. This cytotoxicity requires the type IV secretion system (T4SS). In rough mutants, the cell-surface O antigen is shortened and the T4SS structure is thus exposed on the surface. Cytotoxicity effector proteins can therefore be more easily secreted. This enhanced secretion of effector proteins might cause the increased levels of cytotoxicity observed. However, whether this cytotoxicity is unique to the rough mutant and is mediated by overexpression of the T4SS has not been definitively determined. To test this, in the present study, a virB inactivation mutant (BMΔvirB) and an overexpression strain (BM-VIR) of a smooth Brucella melitensis strain (BM) were constructed and their cytotoxicity for macrophages and intracellular survival capability were analysed and compared. Cytotoxicity was detected in macrophages infected with higher concentrations of strains BM or BM-VIR, but not in those infected with BMΔvirB. The quorum sensing signal molecule N-dodecanoyl-dl-homoserine lactone (C12-HSL), a molecule that can inhibit expression of virB, inhibited the cytotoxicity of BM and BM-VIR, but not of BMΔvirB. These results indicated that overexpression of virB is responsible for Brucella cytotoxicity in macrophages. Transcription analysis showed that virB is regulated in a cell-density-dependent manner both in in vitro culture and during macrophage infection. When compared with BM, BM-VIR showed a reduced survival capacity in macrophages and mice, but both strains demonstrated similar resistance to in vitro stress conditions designed to simulate intracellular environments. Taken together, the cytotoxicity of Brucella for macrophages is probably mediated by increased secretion of effector proteins that results from overexpression of virB or an increase in the number of bacterial cells. The observation that both inactivation and overexpression of virB are detrimental for Brucella intracellular survival also indicated that the expression of virB is tightly regulated in a cell-density-dependent manner.

scholarly journals The AinS autoinducer synthase and LitR master regulator of quorum sensing regulate N-3-hydroxy-decanoyl-homoserine-lactone production and motility in Aliivibrio wodanis 06/09/139

2019 ◽  
Author(s):  
Amudha Deepalakshmi Maharajan ◽  
Hilde Hansen ◽  
Nils Peder Willassen
Keyword(s):  
Quorum Sensing ◽  
Cell Density ◽  
Homoserine Lactone ◽  
Temperature Tolerance ◽  
Dependent Manner ◽  
Secretion Systems ◽  
Master Regulator ◽  
Density Dependent ◽  
Ulcer Disease ◽  
A Cell

Abstract Background Quorum Sensing (QS) is a cell to cell communication system, in which bacteria synthesize and respond to signaling molecules called autoinducers (AI). QS is cell density dependent and known to be involved in regulating virulence, motility and secretion systems to interact with the host or other bacteria. Aliivibrio wodanis is frequently isolated together with Moritella viscosa from the infected Atlantic salmon during outbreaks of the winter ulcer disease. M. viscosa is the main causative agent of the disease while the presence of A. wodanis is still unclear. It is hypothesized that A. wodanis might influence the progression of winter ulcer. The genome of A. wodanis 06/09/139 encodes two autoinducer synthase genes (ainS and luxS) and a master regulator litR. LitR homologs in other aliivibrios have been shown to regulate several phenotypes in a cell density dependent manner. Moreover, a previous study has shown that A. wodanis 06/09/139 produces only one AHL N-3-hydroxy-decanoyl-homoserine-lactone (3OHC10-HSL). Hence, in this work, we have studied the QS system in A. wodanis 06/09/139 by knocking out QS genes ainS and litR. The effects of the deletions were studied with regard to growth, AHL production and motility at different temperatures. Results By using HPLC-MS/MS, we found that the deletion of ainS in A. wodanis 06/09/139 resulted in the loss of 3OHC10-HSL production. The 3OHC10-HSL production in A. wodanis 06/09/139 increased with increase in cell density and more 3OHC10-HSL was produced at 6°C than at 12, 16 and 20°C. The litR mutant demonstrated a ~20% reduction in the production of 3OHC10-HSL relative to the wild type at the stationary phase. Compared to the wildtype and the ainS mutant strains, the litR mutant resulted in a strain with improved temperature tolerance. The motility in mutants (∆litR and ∆ainS) were significantly higher than that of the wildtype. Conclusions Our study shows that AinS in A. wodanis 06/09/139 is the AHL synthase responsible for 3OHC10-HSL production, where the production is both cell density and temperature dependent. Our data also shows that LitR regulates 3OHC10-HSL production only to a minor extent and both LitR and AinS are negative regulators of motility.

scholarly journals Two dynamin-like proteins stabilize FtsZ rings duringStreptomycessporulation

2017 ◽  
Vol 114 (30) ◽  
pp. E6176-E6183 ◽  
Author(s):  
Susan Schlimpert ◽  
Sebastian Wasserstrom ◽  
Govind Chandra ◽  
Maureen J. Bibb ◽  
Kim C. Findlay ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Interactions ◽  
Time Lapse ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Organelle Division ◽  
Z Ring ◽  
A Cell ◽  
Massive Cell

During sporulation, the filamentous bacteriaStreptomycesundergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the twoStreptomycesdynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein–protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

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