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Microtubule Inhibitors Enhance DNA Transfection Efficiency

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Microtubule Inhibitors Enhance DNA Transfection Efficiency bioRxiv preprint doi: https://doi.org/10.1101/2021.05.13.443985; this version posted May 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Microtubule inhibitors enhance DNA transfection 2 efficiency through autophagy receptor p62/SQSTM1 3 4 Megumi Tsuchiya1#, Hidesato Ogawa1#*, Kento Watanabe1, Takako Koujin2, Chie 5 Mori2, Kazuto Nunomura3, Bangzhong Lin3, Akiyoshi Tani3, Yasushi Hiraoka1,2, and 6 Tokuko Haraguchi1,2* 7 8 1Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita 9 565-0871, Japan 10 2Advanced ICT Research Institute Kobe, National Institute of Information and 11 Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe 651-2492, 12 Japan 13 3Graduate School of Pharmaceutical Science, Osaka University, 1-6 Yamadaoka, Suita 14 565-0871, Japan 15 # These authors contributed equally to this manuscript. 16 17 *Correspondence should be addressed to: 18 Hidesato Ogawa, Tel: +81 668794621; Fax: +81 668794622 19 E-mail address: [email protected] 20 Tokuko Haraguchi, Tel: +81 668794621; Fax: +81 668794622 21 E-mail address: [email protected] 22 23 Short title: Tubulin inhibitors enhance transfection efficiency 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.13.443985; this version posted May 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 24 Abstract 25 Ectopic gene expression is an indispensable tool in biology and medicine. However, it 26 is often limited by the low efficiency of DNA transfection. It is known that depletion of 27 p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of 28 transfected DNA. Therefore, p62 is a potential target of drugs to increase transfection 29 efficiency. To identify drugs that enhance transfection efficiency, a non-biased high- 30 throughput screening was applied to over 4,000 compounds from the Osaka University 31 compound library, and their p62-dependency was evaluated. The top-scoring drugs 32 were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them 33 showed positive effects only in the presence of p62. To understand the mechanisms, the 34 time of p62-dependent ubiquitination was examined using polystyrene beads that were 35 introduced into cells as materials that mimicked transfected DNA. The microtubule 36 inhibitors caused a delay in the ubiquitination. Furthermore, the level of phosphorylated 37 p62 at S405, which is required for ubiquitination during autophagosome formation, 38 markedly decreased in the drug-treated cells. These results suggest that microtubule 39 inhibitors inhibit p62-dependent autophagosome formation. Our findings provide new 40 insights into the mechanisms of DNA transfection and also provide a solution to 41 increase DNA transfection efficiency. 42 43 Keywords: high-throughput screening; autophagy; LC3; p62; phosphorylation; 44 ubiquitination; gene delivery 45 46 Abbreviations: ATG, autophagy-related gene; CK2, Casein kinase 2; GAPDH, 47 glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; HdAC6, 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.13.443985; this version posted May 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 48 histone deacetylase 6; KO, knockout; LC3, microtubule-associated protein light chain 3; 49 MEF, murine embryonic fibroblast; NF-κB, nuclear factor-kappa B; TBK1, TANK- 50 binding kinase 1; ULK1, Unc-51 like autophagy activating kinase1; WIPI1, Wd repeat 51 domain phosphoinositide-interacting protein 1 52 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.13.443985; this version posted May 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 Introduction 54 Gene delivery is one of the most important steps for gene therapy and genetic 55 modification in basic science. Gene therapy has great potential in clinical medicine, and 56 this concept has become well established in therapeutic approaches. In basic science, 57 DNA transfection is a powerful tool that enables the study of gene functions and their 58 products in cells. 59 In the gene delivery process, endocytosis is a crucial pathway for the 60 regulation of cellular uptake of plasmid DNA1-3. Importantly, endocytosis can promote 61 the induction of selective autophagy, also called xenophagy4. Generally, autophagy is a 62 cytosolic bulk degradation pathway for recycling biomolecules through nonspecific 63 degradation of proteins and organelles under nutrient starvation conditions. In contrast, 64 selective autophagy plays an important defensive role against cellular infection by 65 pathogens, as part of a starvation-independent autophagic defense system5-7. The 66 conjugation of ubiquitin (Ub) to target pathogens is an initial and important process in 67 selective autophagy. Ubiquitination assists in the recruitment of autophagy receptor 68 proteins, including p62/sequestosome-1 (p62/SQSTM1), against pathogens or 69 transfected DNA4, 8-11. Therefore, suppression of the autophagy pathway can be a target 70 for drugs to increase transfection efficiency. 71 We have reported that the depletion of p62/SQSTM1 protein (hereafter 72 designated p62) greatly increases the efficiency of DNA transfection in cultured cells11. 73 To monitor the behavior of the transfected DNA, we developed an experimental system 74 using DNA-conjugating beads that mimic the transfected DNA. In this system, DNA- 75 conjugated beads together with pHrodo, a fluorescent marker for endosome rupture, are 76 incorporated into cells and their intracellular dynamics are analyzed in a live cell11. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.13.443985; this version posted May 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 77 Using this system, we demonstrated that the transfected DNA is incorporated into cells 78 through endocytosis, released into the cytosol from the endosomes, and entrapped via 79 autophagy in a p62-dependent manner. Furthermore, we demonstrated that the 80 recruitment of Ub around the transfected material is significantly delayed in p62 gene- 81 knockout murine embryonic fibroblast (p62KO-MEF) cells compared with that in 82 normal MEF cells12. Additionally, the phosphorylation of S405 (human S403) of p62 is 83 a crucial step for the recruitment of Ub to the target site of transfected materials that 84 mimic ectopic DNA11. Hence, phosphorylation of S405 of p62 is an essential step for 85 transfection-induced selective autophagy12. Since p62 plays an important role in gene 86 delivery through initial ubiquitination of the transfected DNA, inhibition of p62 may 87 increase transfection efficiency. The aim of this study was to identify a small chemical 88 compound that blocks the initial step of p62-dependent selective autophagy to enhance 89 dNA transfection efficiency in mammalian cells. 90 91 Results 92 High-throughput screening for drugs enhancing transfection efficiency 93 To identify potential compounds that can enhance transfection efficiency, high- 94 throughput screening based on a luciferase assay was performed on MEF cells using an 95 automated workstation. MEF cells were seeded in 384-well plates and incubated for 18 96 h with each compound from the Osaka University compound library (4,400 97 compounds)13, at a final concentration of 10 µM. As a negative control, dMSO (at a 98 final concentration of 1%) was used instead of the compounds. The cells were 99 transfected with the pCMV-Luc plasmid and incubated for 28 h; luciferase gene 100 expression was driven by the cytomegalovirus immediate early (CMV-IE) promoter in 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.13.443985; this version posted May 13, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 101 this plasmid. After cell viability assay, a luciferase reporter assay was carried out (Fig. 102 1A). Among the 4,400 compounds tested, 160 had severe effects on cell viability and 103 were therefore removed from further analysis. For the remaining 4,240 compounds, cell 104 viability in the presence of each compound was more than 96%. The transfection 105 efficiency of the cells treated with each compound is plotted in Fig. 1B, the luciferase 106 activity was normalized to cell viability. In this first screening, out of the 4,240 tested 107 compounds, we identified 87 compounds that increased luciferase activity compared 108 with the negative control (approximately 2.1% positive hit rate). The cutoff value used 109 for selection was the mean value of the DMSO control + 4 × standard deviation (SD) 110 (mean 0.347, Sd = 0.242). 111 The activity of these 87 compounds was further analyzed via a second 112 screening using MEF cells cultured in 96-well plates and incubated for 16 h with each 113 of these compounds at a concentration of 1 μM. The luciferase reporter assay showed 114 that the transfection efficiency increased in the presence of each of these compounds, 115 with a range of approximately 2- to 260-fold that of the control, dMSO (Fig.
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