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

Ana E Gomez Martinez,Amy E Herr

doi:10.1021/acsmeasuresciau.1c00014

Ana E Gomez Martinez, Amy E Herr

Open Access

https://doi.org/10.1021/acsmeasuresciau.1c00014

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Abstract

Gaining insight into the timing of cell apoptosis events requires single-cell-resolution measurements of cell viability. We explore the supposition that mechanism-based scrutiny of programmed cell death would benefit from same-cell analysis of both the DNA state (intact vs fragmented) and the protein states, specifically the full-length vs cleaved state of the DNA-repair protein PARP1, which is cleaved by caspase-3 during caspase-dependent apoptosis. To make this same-cell, multimode measurement, we introduce the single-cell electrophoresis-based viability and protein (SEVAP) assay. Using SEVAP, we (1) isolate human breast cancer SKBR3 cells in microwells molded in thin polyacrylamide gels, (2) electrophoretically separate protein molecular states and DNA molecular states—using differences in electrophoretic mobility—from each single-cell lysate, and (3) perform in-gel DNA staining and PARP1 immunoprobing. Performed in an open microfluidic device, SEVAP scrutinized hundreds to thousands of individual SKBR3 cells. In each single-cell lysate separation, SEVAP baseline-resolved fragmented DNA from intact DNA (Rs = 5.17) as well as cleaved PARP1 from full-length PARP1 (Rs = 0.66). Comparing apoptotic and viable cells showed statistically similar profiles (expression, mobility, peak width) of housekeeping protein β-tubulin (Mann–Whitney U test). Clustering and cross-correlation analysis of DNA migration and PARP1 migration identified nonapoptotic vs apoptotic cells. Clustering analysis further suggested that cleaved PARP1 is a suitable apoptosis marker for this system. SEVAP is an efficient, multimode, end-point assay designed to elucidate cell-to-cell heterogeneity in mechanism-specific signaling during programmed cell death.

Highlights

Dysregulated apoptosis mechanisms contribute to neurodegenerative disorders,[1] cancers,[1,2] and chemotherapeutic resistance.[3]
For the multimode single-cell assay, we start by developing a single-cell proteoform separation mode to distinguish fulllength versus cleaved PARP1
We use knowledge acquired from that most-stringent molecular analysis (PARP1 forms) to design and develop the less-stringent, coarse DNA fractionation polyacrylamide gel electrophoresis (PAGE) mode

Summary

Introduction

Dysregulated apoptosis mechanisms contribute to neurodegenerative disorders,[1] cancers,[1,2] and chemotherapeutic resistance.[3] Understanding cancer-cell-death avoidance and chemotherapy resistance to determine drug targets requires scrutiny of programmed cell-death pathways, including apoptosis.[3] During apoptosis, the cellular membrane blebs, DNA condenses, the caspase cascade modifies proteins, and DNA is fragmented into ∼50 kb fragments by active DNases.[4]. Accurate temporal resolution of apoptotic events requires single-cell resolution[5] due to the asynchronous nature of apoptosis.[2] The gold standard measurement of apoptosis is morphological changes (membrane blebbing, nuclear fragmentation). Other common measurements are imaging- or flowcytometry-based analyses of stains.[2] These stains penetrate a compromised cell membrane (e.g., ethidium homodimer-1, propidium iodide, SYTOX Green) or detect externalized phosphatidylserine (Annexin V).[2]

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