High-Throughput Screening to Identify Proteins Involved in Nanopores Formation After nsPEF
Speaker Biographical Sketch
Giedre Silkuniene worked as a Junior Researcher at the Lithuanian University of Health Sciences, Kaunas, Lithuania. Primary research: investigation of digestive system tumors, their response to chemotherapy treatment, and treatment efficiency enhancement. In 2020 Giedre received a PhD in Biochemistry (Thesis: The Effect of Hyperthermia and Hyperthermic Chemotherapy on Cells, Mitochondria and Their Enzymes) at Vytautas Magnus University, Kaunas, Lithuania. Since 2020 works as a Postdoctoral Research Assistant at Old Dominion University, Norfolk, United States. The main postdoctoral research aims to identify membrane protein targets for nsPEF-induced pore formation.
2021 Frank Reidy Research Center for Bioelectrics Retreat
It is widely accepted that plasma membrane permeabilization after PEF occurs due to hydrophilic pores formation in the lipid bilayer. The concept of pore formation is confirmed by molecular dynamics (MD) simulations. However, there is increasing evidence that PEF can cause not only hydrophilic pore formation but also modulate membrane proteins and their function, and these changes could be part of increased membrane’s permeability after PEF. Ion channels, especially voltage-gated ion channels, are probable targets of PEF. However, ion channels are an extremely large group, and to identify specific channels responsible for increased conductivity after PEF, high-throughput screening is needed. In this work, we used the lentiviral CRISPR/Cas9 library designed for knockout (KO) of 329 different ion channels or other membrane-associated proteins in the U937/Cas9 cell model. After applying nsPEF (300ns, 7 kV/cm, 20 or 40 pulses at 20 Hz), each KO was screened for YoPro-1 uptake using a custom assembled screening station based on an inverted microscope equipped with automated stage repositioning, IR laser hardware autofocusing (ZDC), and image stitching capabilities. Out of 329 screened KOs, 9 showed lower YO-PRO-1 uptake than control. Such results indicate that proteins coded by these genes either support molecular transport across nsPEF stimulated membrane or proteins could be directly damaged by nsPEF. 20 KOs responded to nsPEF with higher YO-PRO-1 uptake than control, suggesting that these proteins may play the regulatory function of ion channels, or they could be linked to membrane stability and/or repair of the membrane after the electric injury. This is the first high-throughput screening to identify possible nsPEF protein targets in the cell plasma membrane.
Silkuniene, Giedre; Mangalanathan, Uma Maheswari; Silkunas, Mantas; Pakhomov, Andrei G.; and Pakhomova, Olga N., "High-Throughput Screening to Identify Proteins Involved in Nanopores Formation After nsPEF" (2021). 2021 Frank Reidy Research Center for Bioelectrics Retreat. 9.
0000-0003-3816-3860 (Pakhomov); 0000-0003-4950-4130 (Pakhomova)