Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases Anandika Dhaliwal1, Maricela Maldonado 2 , Clayton Lin2, Tatiana Segura1,2* 1 Biomedical Engineering Interdepartmental Program, University of California Los Angeles, Los Angeles, California, United States of America, 2 Chemical and Biomolecular Engineering Department, University of California Los Angeles, Los Angeles, California, United States of America Abstract Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer. Citation: Dhaliwal A, Maldonado M, Lin C, Segura T (2012) Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases. PLoS ONE 7(4): e35046. doi:10.1371/journal.pone.0035046 Editor: Neil A. Hotchin, University of Birmingham, United Kingdom Received November 30, 2011; Accepted March 12, 2012; Published April 11, 2012 Copyright: 2012 Dhaliwal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by National Institutes of Health R21EB009516, Understanding the Role of the Extracellular Matrix on Non-Viral Gene Transfer (www.nih.gov). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tsegura@ucla.edu Introduction Although gene delivery can be a robust approach to treat disease and augment tissue formation, limitations with effective and save delivery have limited its success as a therapy. Gene delivery to mammalian cells can be achieved using viral as well as non-viral delivery systems [1]. Non-viral gene delivery systems have the advantage of being less immunogenic compared to viral gene delivery systems as well as allow for large-scale production and modularity. However, they are limited by their efficacy. Previous studies have focused on engineering more efficient delivery vehicles that can overcome one or more of the barriers to efficient gene transfer [2,3]. Although less common, recent studies have looked at the cellular microenvironment and the cell itself to elucidate other approaches to enhance non-viral gene transfer [4– 8]. For example, the stiffness of the matrix where the cells are plated modulates non-viral gene delivery with cells plated on stiff surfaces (110 KPa) resulting in enhanced gene transfer due to increased cell proliferation and survival [8]. Collagen I and IV have been shown to enhance gene transfer in PC12 cells, which was correlated with the relative projected nuclear area of the plated cells [9], while fibronectin and collagen I have been shown to enhance gene expression in NIH/3T3 cells which has been correlated to the relative increased internalization on these surfaces and the pathway of internalization [11]. Cationic lipid- mediated gene transfer to rat smooth muscle cells is enhanced when the cells are plated on surfaces that promote avß3 binding, with antibodies against avß3 and ß3 decreasing the amount of gene transfer [9]. Further, our laboratory has shown that cellular microenvironment modulates non-viral gene delivery to mouse mesenchymal stem cells (mMSCs), specifically we screened 6 different ECM proteins and their combinations for their ability to enhance gene transfer to mouse mesenchymal stem cells (mMSCs) using poly(ethylene imine) polyplexes. We found that proteins that promoted well spread cells (e.g. fibronectin and collagen IV) resulted in polyplexes being trafficked to the nucleus and enhanced gene transfer, while those that resulted in less spread cells (e.g. collagen I) resulted in polyplexes that did not colocalize with the nucleus and inhibited gene transfer [10]. When comparing the internalization pathway of polyplexes for cells seeded on fibronectin or collagen I, we found that different endocytic pathways are used, with clathrin-mediated endocytosis being the primary pathway used for cells plated on fibronectin [6]. Further, polymerized actin, actin-myosin interactions, and the microtubu- lar network were found to influence non-viral gene transfer to different extends for cells seeded on fibronectin versus collagen I [6]. Structural components of the ECM such as Fn are able to actively mediate crosstalk between the ECM and RhoGTPases by associating with cell surface receptors, namely integrins [11] and PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35046