Abstract
Objectives
During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection.
Results
A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation.
Conclusions
Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The data that support the findings of the current study are available from the corresponding author on request.
References
Casella JF, Flanagan MD, Lin S (1981) Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature 293:302–305. https://doi.org/10.1038/293302a0
Chan CJ, Ekpenyong AE, Golfier S, Li W, Chalut KJ, Otto O, Elgeti J, Guck J, Lautenschläger F (2015) Myosin II activity softens cells in suspension. Biophys J 108:1856–1869. https://doi.org/10.1016/j.bpj.2015.03.009
Clausen MP, Colin-York H, Schneider F, Eggeling C, Fritzsche M (2017) Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy. J Phys D 50:064002. https://doi.org/10.1088/1361-6463/aa52a1
Coué M, Brenner SL, Spector I, Korn ED (1987) Inhibition of actin polymerization by latrunculin A. FEBS Lett 213:316–318. https://doi.org/10.1016/0014-5793(87)81513-2
Dinic J, Ashrafzadeh P, Parmryd I (2013) Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains. Biochim Biophys Acta Biomembr 1828:1102–1111. https://doi.org/10.1016/j.bbamem.2012.12.004
Dutta D, Asmar A, Stacey M (2015) Effects of nanosecond pulse electric fields on cellular elasticity. Micron 72:15–20. https://doi.org/10.1016/j.micron.2015.01.004
Flormann DAD, Kaub KH, Vesperini D, Schu M, Anton C, Pohland MO, Kainka L, Montalvo Bereau G, Janshoff A, Hawkins RJ, Terriac E, Lautenschläger F (2021) The role of actin and myosin II in the cell cortex of adhered and suspended cells. bioRxiv. https://doi.org/10.1101/2021.08.03.454901
Haberl S, Kandušer M, Flisar K, Hodžić D, Bregar VB, Miklavčič D, Escoffre JM, Rols MP, Pavlin M (2013) Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level. J Gene Med 15:169–181. https://doi.org/10.1002/jgm.2706
He S, Singh D, Helfield B (2022) An overview of cell membrane perforation and resealing mechanisms for localized drug delivery. Pharmaceutics 14:886. https://doi.org/10.3390/pharmaceutics14040886
Horn A, Jaiswal JK (2018) Cellular mechanisms and signals that coordinate plasma membrane repair. Cell Mol Life Sci 75:3751–3770. https://doi.org/10.1007/s00018-018-2888-7
Huang W, Matsui TS, Saito T, Kuragano M, Takahashi M, Kawahara T, Sato M, Deguchi S (2021) Mechanosensitive myosin II but not cofilin primarily contributes to cyclic cell stretch-induced selective disassembly of actin stress fibers. Am J Physiol, Cell Physiol 320:C1153–C1163. https://doi.org/10.1152/ajpcell.00225.2020
Huang W, Sakuma S, Tottori N, Sugano SS, Yamanishi Y (2022) Viscosity-aided electromechanical poration of cells for transfecting molecules. Lab Chip 22:4276–4291. https://doi.org/10.1039/D2LC00628F
Ichikawa K, Maeda S, Yamanishi Y (2018) Evaluation of invasiveness by breakdown phenomena of electrically induced bubbles for a needle-free injector. J Microelectromech Syst 27:305–311. https://doi.org/10.1109/JMEMS.2018.2793314
Kim TK, Eberwine JH (2010) Mammalian cell transfection: the present and the future. Anal Bioanal Chem 397:3173–3178. https://doi.org/10.1007/s00216-010-3821-6
Lam JKW, Armes SP, Stolnik S (2011) The involvement of microtubules and actin filaments in the intracellular transport of non-viral gene delivery system. J Drug Target 19:56–66. https://doi.org/10.3109/10611861003733938
Matsui TS, Kaunas R, Kanzaki M, Sato M, Deguchi S (2011) Non-muscle myosin II induces disassembly of actin stress fibres independently of myosin light chain dephosphorylation. Interface Focus 1:754–766. https://doi.org/10.1098/rsfs.2011.0031
Meulenberg CJ, Todorovic V, Cemazar M (2012) Differential cellular effects of electroporation and electrochemotherapy in monolayers of human microvascular endothelial cells. PLoS ONE 7:e52713. https://doi.org/10.1371/journal.pone.0052713
Mortensen K, Larsson LI (2003) Effects of cytochalasin D on the actin cytoskeleton: association of neoformed actin aggregates with proteins involved in signaling and endocytosis. Cell Mol Life Sci 60:1007–1012. https://doi.org/10.1007/s00018-003-3022-x
Muralidharan A, Rems L, Kreutzer M, Boukany P (2020) Actin networks regulate the cell membrane permeability during electroporation. Biochim Biophys Acta Biomembr 1863:183468. https://doi.org/10.1016/j.bbamem.2020.183468
Pehlivanova VN, Tsoneva IH, Tzoneva RD (2012) Multiple effects of electroporation on the adhesive behaviour of breast cancer cells and fibroblasts. Cancer Cell Int 12:9. https://doi.org/10.1186/1475-2867-12-9
Perrier DL, Vahid A, Kathavi V, Stam L, Rems L, Mulla Y, Muralidharan A, Koenderink GH, Kreutzer MT, Boukany PE (2019) Response of an actin network in vesicles under electric pulses. Sci Rep 9:8151. https://doi.org/10.1038/s41598-019-44613-5
Rols M-P, Teissié J (1992) Experimental evidence for the involvement of the cytoskeleton in mammalian cell electropermeabilization. Biochim Biophys Acta Biomembr 1111:45–50. https://doi.org/10.1016/0005-2736(92)90272-N
Rotsch C, Radmacher M (2000) Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study. Biophys J 78:520–535. https://doi.org/10.1016/s0006-3495(00)76614-8
Sawatsubashi S, Joko Y, Fukumoto S, Matsumoto T, Sugano SS (2018) Development of versatile non-homologous end joining-based knock-in module for genome editing. Sci Rep 8:593. https://doi.org/10.1038/s41598-017-18911-9
Steuer A, Schmidt A, Labohá P, Babica P, Kolb JF (2016) Transient suppression of gap junctional intercellular communication after exposure to 100-nanosecond pulsed electric fields. Bioelectrochemistry 112:33–46. https://doi.org/10.1016/j.bioelechem.2016.07.003
Steuer A, Wende K, Babica P, Kolb JF (2017) Elasticity and tumorigenic characteristics of cells in a monolayer after nanosecond pulsed electric field exposure. Eur Biophys J 46:567–580. https://doi.org/10.1007/s00249-017-1205-y
Svitkina TM (2020) Actin cell cortex: structure and molecular organization. Trends Cell Biol 30:556–565. https://doi.org/10.1016/j.tcb.2020.03.005
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. https://doi.org/10.1016/j.cell.2006.07.024
Ujihara Y, Miyazaki H, Wada S (2008) Morphological study of fibroblasts treated with cytochalasin D and colchicine using a confocal laser scanning microscopy. J Physiol Sci 58(7):499–506
Wakatsuki T, Schwab B, Thompson NC, Elson EL (2001) Effects of cytochalasin D and latrunculin B on mechanical properties of cells. J Cell Sci 114:1025–1036. https://doi.org/10.1242/jcs.114.5.1025
Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398. https://doi.org/10.1038/nbt1026
Xiao D, Tang L, Zeng C, Wang J, Luo X, Yao C, Sun C (2011) Effect of actin cytoskeleton disruption on electric pulse-induced apoptosis and electroporation in tumour cells. Cell Biol Int 35:99–104. https://doi.org/10.1042/cbi20100464
Zhou ZL, Sun XX, Ma J, Man CH, Wong AST, Leung AY, Ngan AHW (2016) Mechanical oscillations enhance gene delivery into suspended cells. Sci Rep 6:22824. https://doi.org/10.1038/srep22824
Acknowledgements
The authors are grateful to Dr. H. Matsubayashi for his advice of actin filament. The authors are also grateful to Dr. Shigeo Sugano for his advice of analysis of our results. We thank Frank Kitching, MSc, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
Supporting information
Movie S1—The bubble injector and the growth, ejection, and collapse of microbubbles. The movie was obtained using a Hyper Vision HPV-X2 high-speed video camera (Shimadzu).
Movie S2—Microbubbles at a cell concentration of 2.1 × 105 cells/μL (NIH/3T3) (high cell concentration). The movie was obtained using a Hyper Vision HPV-X2 high-speed video camera (Shimadzu).
Funding
This work was supported by Core Research for Evolutional Science and Technology (CREST) grant from the Japan Science and Technology Agency (JST) (grant number: JPMJCR19S6) and JST Moonshot R&D (JPMJMS2217-3-1).
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by WH, YM, and YY. The first draft of the manuscript was written by WH and NT. All authors revised each version of the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The patents related to this work were filed on 17 September 2020 (PCT/JP2020/035332) and on 31 August 2022 (QP210075).
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary file1 (AVI 7912 KB)
The bubble injector and the growth, ejection, and collapse of microbubbles. The movie was obtained using a Hyper Vision HPV-X2 high-speed video camera (Shimadzu).
Supplementary file2 (AVI 9369 KB)
Microbubbles at a cell concentration of 2.1 × 105 cells/μL (NIH/3T3) (high cell concentration). The movie was obtained using a Hyper Vision HPV-X2 high-speed video camera (Shimadzu).
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Huang, W., Ma, Y., Tottori, N. et al. Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation. Biotechnol Lett 45, 1417–1430 (2023). https://doi.org/10.1007/s10529-023-03382-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-023-03382-y