ID: 2173

• Title:
  Contribution of Membrane Proteins to Nanosecond Electric Pulse-Induced Membrane Disruption and Recovery
  
  Authors:
  

  Silkuniene, Giedre- FRRCBE, ODU, Norfolk, VA USA; Lithuanian University of Health Sciences, Kaunas, Lithuania
  Mangalanathan, Uma - FRRCBE, ODU, Norfolk, VA USA 
  Rossi, Alessandra - FRRCBE, ODU, Norfolk, VA USA; Sapienza University of Rome, Italy 
  Pakhomov, Andrei - FRRCBE, ODU, Norfolk, VA USA  
  Pakhomova, Olga- FRRCBE, ODU, Norfolk, VA USA

  
  
  Abstract:
  The electropermeabilized state in cells lasts orders of magnitude longer than in lipid vesicles and molecular models of lipid membranes. This difference can potentially be attributed to the complexity of cell membranes, including the presence of the cytoskeleton and a large fraction of diverse membrane proteins. Our study aimed to identify membrane proteins which assist or impede electroporation by nanosecond electric pulses (nsEP).
  
  We used a LentiArray CRISPR Ion Channel library to knockout (KO) genes in U937 human monocytes stably expressing Cas9 nuclease . We generated a total of 316 U937 cell derivatives, each with a KO for a single gene. Each KO was treated in electroporation cuvettes with trains of 20 or 40 pulses (300 ns, 7 kV/cm, 20Hz) in the presence of a membrane-impermeable fluorescent dye, Yo-Pro-1 (YP). YP entry due to electroporation was measured using an Olympus IX83 microscope configured for high-throughput screening. We scanned nine fields of view in each well and stitched them into one image, yielding 400-2,000 cells per sample. We quantified YP fluorescence in individual cells with the Advanced CellScoring package of MetaMorph and averaged YP emission intensity across all cells identified in each sample. For each KO variant, we calculated the difference in YP uptake between nsEP_ and sham-exposed samples, and this difference was further compared controls transduced with a scrambled (non-targeting) gRNA. Significance of differences was assessed using Dunnett’s test, which compares multiple groups with a single control. we also characterized the magnitude of suspected effects using the strictly standardized mean difference (SSMD), a commonly used criterion in high-throughput screening.
  
  We found that many more KOs resulted in YP uptake increase than in its reduction. Specifically, the number of KOs with the mean YP uptake above 110% and 120% was 135 and 65, respectively, compared to only 50 below 90% and 16 below 80%. Additionally, the median YP uptake showed an unequal distribution, with a 2- to 4-fold larger number of KOs with increased YP uptake (e.g., 58 KOs >110% but only 15 KOs <90%). The predominance of the YP increase trend is underscored by the fact that SSMD exceeded 1.0 in as many as 28 KOs but fell below (-1.0) in only 5 KOs. Furthermore, the difference from SCR control was statistically significant at p<0.05 in 17 KOs with increased YP uptake, but not in a single KO with decreased YP uptake. This response pattern suggests that KO of many different genes can make the cell membrane more vulnerable to nsEP by altering its resilience or repair mechanisms, whereas KOs that reduce membrane permeabilization by nsEP are few or nonexistent.
  
  
  
  In the subsequent series of verification screenings, only two KOs, for the SCNN1A and CLCA1 genes, showed a statistically significant reduction in YP uptake. It is possible that the respective proteins are part of electropermeabilization lesions or that they increase the lifespan of such lesions. On the other hand, as many as 39 genes were identified as likely hits for the increased YP uptake, indicating that the respective proteins contributed to membrane stability or repair after nsEP. Additionally, the expression level of eight genes in different human cell types showed a strong correlation (R>0.9, p<0.02) with their LD50 for lethal nsEP treatments. Therefore, these genes could serve as a criterion for evaluating the selectivity and efficiency of hyperplasia ablations using nsEP.
  
  
  
  Keywords:
  Membrane lesions; membrane repair, membrane proteins,ion channels; nsPEF; electroporation; electropermeabilization;
  
  Refs:
  
  
  Topic 1:
  1. Biological responses (molecular, subcellular, cellular and intercellular)
  
  Topic 2:
  2. Biophysics and biochemistry of interaction mechanisms