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Related Concept Videos

Overview of Cell Death01:30

Overview of Cell Death

Cell death is an essential process where the body gets rid of old or damaged cells. Cell proliferation and death need to be balanced, as an imbalance between the two may lead to cancer or autoimmune diseases.
Cell death was observed in the early 19th century, but there was no experimental evidence to prove it. In 1842, Carl Vogt first discovered cell death in a metamorphic toad; however, it was not termed ‘cell death.’ Scientists discovered different cell death pathways only in the 20th century...

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Programmed Cell-Death Mechanism Analysis Using Same-Cell, Multimode DNA and Proteoform Electrophoresis.

Ana E Gomez Martinez1,2, Amy E Herr1,2,3

  • 1Department of Bioengineering, University of California Berkeley, Berkeley, California 94720, United States.

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|December 23, 2021
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We developed the single-cell electrophoresis-based viability and protein (SEVAP) assay to analyze DNA and protein states simultaneously in single cells undergoing apoptosis. This method distinguishes apoptotic from viable cells by assessing DNA fragmentation and PARP1 cleavage, offering insights into programmed cell death heterogeneity.

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Understanding the precise timing of apoptosis requires single-cell resolution of cell viability.
  • Mechanism-based studies of programmed cell death benefit from analyzing both DNA integrity and protein cleavage states within the same cell.
  • Caspase-3 cleavage of PARP1 is a key event in caspase-dependent apoptosis.

Purpose of the Study:

  • To introduce and validate the single-cell electrophoresis-based viability and protein (SEVAP) assay for simultaneous, same-cell analysis of DNA and protein states.
  • To assess the utility of SEVAP in distinguishing between apoptotic and viable human breast cancer cells.
  • To investigate cell-to-cell heterogeneity in programmed cell death signaling.

Main Methods:

  • Development of the SEVAP assay using microfluidic devices with polyacrylamide gels for in-gel electrophoresis.
  • Isolation of individual SKBR3 cells in microwells for single-cell lysate preparation.
  • Electrophoretic separation of molecular states (DNA and protein) based on mobility, followed by in-gel DNA staining and PARP1 immunoprobing.

Main Results:

  • SEVAP successfully resolved fragmented DNA from intact DNA (Rs = 5.17) and cleaved PARP1 from full-length PARP1 (Rs = 0.66) within single-cell lysates.
  • Clustering and cross-correlation analyses of DNA and PARP1 migration patterns effectively identified nonapoptotic versus apoptotic cells.
  • Cleaved PARP1 was identified as a suitable marker for apoptosis detection in this system, with housekeeping protein profiles remaining consistent across cell states.

Conclusions:

  • The SEVAP assay provides an efficient, multimode, end-point method for analyzing cell viability and protein states at the single-cell level.
  • SEVAP enables the elucidation of cell-to-cell heterogeneity in mechanism-specific signaling during programmed cell death.
  • This assay is valuable for detailed investigations into the dynamics of apoptosis in various biological contexts.