Cell Growth and Cell Death Studied by Electric Cell-Substrate Impedance Sensing

AbstractMitochondrial proteases are key components in mitochondrial stress responses that maintain proteostasis and mitochondrial integrity in harsh environmental conditions, which leads to the acquisition of aggressive phenotypes, including chemoresistance and metastasis. However, the molecular mechanisms and exact role of mitochondrial proteases in cancer remain largely unexplored. Here, we identified functional crosstalk between LONP1 and ClpP, which are two mitochondrial matrix proteases that cooperate to attenuate proteotoxic stress and protect mitochondrial functions for cancer cell survival. LONP1 and ClpP genes closely localized on chromosome 19 and were co-expressed at high levels in most human cancers. Depletion of both genes synergistically attenuated cancer cell growth and induced cell death due to impaired mitochondrial functions and increased oxidative stress. Using mitochondrial matrix proteomic analysis with an engineered peroxidase (APEX)-mediated proximity biotinylation method, we identified the specific target substrates of these proteases, which were crucial components of mitochondrial functions, including oxidative phosphorylation, the TCA cycle, and amino acid and lipid metabolism. Furthermore, we found that LONP1 and ClpP shared many substrates, including serine hydroxymethyltransferase 2 (SHMT2). Inhibition of both LONP1 and ClpP additively increased the amount of unfolded SHMT2 protein and enhanced sensitivity to SHMT2 inhibitor, resulting in significantly reduced cell growth and increased cell death under metabolic stress. Additionally, prostate cancer patients with higher LONP1 and ClpP expression exhibited poorer survival. These results suggest that interventions targeting the mitochondrial proteostasis network via LONP1 and ClpP could be potential therapeutic strategies for cancer.

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Electric Cell-Substrate Impedance Sensing To Monitor Viral Growth and Study Cellular Responses to Infection with Alphaherpesviruses in Real Time

mSphere ◽

10.1128/msphere.00039-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 13

Author(s):

Matthew R. Pennington ◽

Gerlinde R. Van de Walle

Keyword(s):

Real Time ◽

Cellular Response ◽

Cellular Responses ◽

Cellular Damage ◽

Impedance Sensing ◽

Novel Technologies ◽

Antiviral Therapies ◽

Mucosal Surfaces ◽

Cell Substrate ◽

Hsv 1

ABSTRACT Alphaherpesviruses, including those that commonly infect humans, such as HSV-1 and HSV-2, typically infect and cause cellular damage to epithelial cells at mucosal surfaces, leading to disease. The development of novel technologies to study the cellular responses to infection may allow a more complete understanding of virus replication and the creation of novel antiviral therapies. This study demonstrates the use of ECIS to study various aspects of herpesvirus biology, with a specific focus on changes in cellular morphology as a result of infection. We conclude that ECIS represents a valuable new tool with which to study alphaherpesvirus infections in real time and in an objective and reproducible manner. Electric cell-substrate impedance sensing (ECIS) measures changes in an electrical circuit formed in a culture dish. As cells grow over a gold electrode, they block the flow of electricity and this is read as an increase in electrical impedance in the circuit. ECIS has previously been used in a variety of applications to study cell growth, migration, and behavior in response to stimuli in real time and without the need for cellular labels. Here, we demonstrate that ECIS is also a valuable tool with which to study infection by alphaherpesviruses. To this end, we used ECIS to study the kinetics of cells infected with felid herpesvirus type 1 (FHV-1), a close relative of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, and compared the results to those obtained with conventional infectivity assays. First, we demonstrated that ECIS can easily distinguish between wells of cells infected with different amounts of FHV-1 and provides information about the cellular response to infection. Second, we found ECIS useful in identifying differences between the replication kinetics of recombinant DsRed Express2-labeled FHV-1, created via CRISPR/Cas9 genome engineering, and wild-type FHV-1. Finally, we demonstrated that ECIS can accurately determine the half-maximal effective concentration of antivirals. Collectively, our data show that ECIS, in conjunction with current methodologies, is a powerful tool that can be used to monitor viral growth and study the cellular response to alphaherpesvirus infection. IMPORTANCE Alphaherpesviruses, including those that commonly infect humans, such as HSV-1 and HSV-2, typically infect and cause cellular damage to epithelial cells at mucosal surfaces, leading to disease. The development of novel technologies to study the cellular responses to infection may allow a more complete understanding of virus replication and the creation of novel antiviral therapies. This study demonstrates the use of ECIS to study various aspects of herpesvirus biology, with a specific focus on changes in cellular morphology as a result of infection. We conclude that ECIS represents a valuable new tool with which to study alphaherpesvirus infections in real time and in an objective and reproducible manner.

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Lipid biomarkers of glioma cell growth arrest and cell death detected by 1 H magic angle spinning MRS

NMR in Biomedicine ◽

10.1002/nbm.2796 ◽

2012 ◽

Vol 25 (11) ◽

pp. 1253-1262 ◽

Cited By ~ 19

Author(s):

Ladan Mirbahai ◽

Martin Wilson ◽

Christopher S. Shaw ◽

Carmel McConville ◽

Roger D. G. Malcomson ◽

...

Keyword(s):

Cell Death ◽

Cell Growth ◽

Glioma Cell ◽

Growth Arrest ◽

Magic Angle Spinning ◽

Lipid Biomarkers ◽

Magic Angle ◽

Cell Growth Arrest ◽

Angle Spinning

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Electrical cell-substrate impedance sensing as a non-invasive tool for cancer cell study

The Analyst ◽

10.1039/c0an00560f ◽

2011 ◽

Vol 136 (2) ◽

pp. 237-245 ◽

Cited By ~ 109

Author(s):

Jongin Hong ◽

Karthikeyan Kandasamy ◽

Mohana Marimuthu ◽

Cheol Soo Choi ◽

Sanghyo Kim

Keyword(s):

Cancer Cell ◽

Impedance Sensing ◽

Non Invasive ◽

Cell Substrate ◽

Cell Study

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Comparison of Leading Biosensor Technologies to Detect Changes in Human Endothelial Barrier Properties in Response to Pro-Inflammatory TNFα and IL1β in Real-Time

Biosensors ◽

10.3390/bios11050159 ◽

2021 ◽

Vol 11 (5) ◽

pp. 159

Author(s):

James J. W. Hucklesby ◽

Akshata Anchan ◽

Simon J. O'Carroll ◽

Charles P. Unsworth ◽

E. Scott Graham ◽

...

Keyword(s):

Human Brain ◽

Real Time ◽

Barrier Properties ◽

Endothelial Barrier ◽

Endothelial Monolayer ◽

Impedance Sensing ◽

Endothelial Monolayers ◽

Impedance Data ◽

Cell Substrate ◽

Limited Frequency

Electric Cell-Substrate Impedance Sensing (ECIS), xCELLigence and cellZscope are commercially available instruments that measure the impedance of cellular monolayers. Despite widespread use of these systems individually, direct comparisons between these platforms have not been published. To compare these instruments, the responses of human brain endothelial monolayers to TNFα and IL1β were measured on all three platforms simultaneously. All instruments detected transient changes in impedance in response to the cytokines, although the response magnitude varied, with ECIS being the most sensitive. ECIS and cellZscope were also able to attribute responses to particular endothelial barrier components by modelling the multifrequency impedance data acquired by these instruments; in contrast the limited frequency xCELLigence data cannot be modelled. Consistent with its superior impedance sensing, ECIS exhibited a greater capacity than cellZscope to distinguish between subtle changes in modelled endothelial monolayer properties. The reduced resolving ability of the cellZscope platform may be due to its electrode configuration, which is necessary to allow access to the basolateral compartment, an important advantage of this instrument. Collectively, this work demonstrates that instruments must be carefully selected to ensure they are appropriate for the experimental questions being asked when assessing endothelial barrier properties.

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