title: Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases.
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.
authors.affiliation: Biomedical Engineering Interdepartmental Program, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Anandika
authors.initials: A
authors.lastname: Dhaliwal
authors.forename: Maricela
authors.initials: M
authors.lastname: Maldonado
authors.forename: Clayton
authors.initials: C
authors.lastname: Lin
authors.forename: Tatiana
authors.initials: T
authors.lastname: Segura
mesh_terms: D036002:ADP Ribose Transferases / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology; D001905:Botulinum Toxins / Q000494:pharmacology; D002460:Cell Line; D060833:Cellular Microenvironment; D003599:Cytoskeleton / Q000378:metabolism*; D004705:Endocytosis; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019076:Transgenes / Q000235:genetics*; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D009479:Neuropeptides; C484602:Rac1 protein, mouse; C031815:toxB protein, Clostridium difficile; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; C526380:RhoA protein, mouse; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein