title: Stimulation of in vivo nuclear transport dynamics of actin and its co-factors IQGAP1 and Rac1 in response to DNA replication stress.
abstract: Actin, a constituent of the cytoskeleton, is now recognized to function in the nucleus in gene transcription, chromatin remodeling and DNA replication/repair. Actin shuttles in and out of the nucleus through the action of transport receptors importin-9 and exportin-6. Here we have addressed the impact of cell cycle progression and DNA replication stress on actin nuclear localization, through study of actin dynamics in living cells. First, we showed that thymidine-induced G1/S phase cell cycle arrest increased the nuclear levels of actin and of two factors that stimulate actin polymerization: IQGAP1 and Rac1 GTPase. When cells were exposed to hydroxyurea to induce DNA replication stress, the nuclear localization of actin and its regulators was further enhanced. We employed live cell photobleaching assays and discovered that in response to DNA replication stress, GFP-actin nuclear import and export rates increased by up to 250%. The rate of import was twice as fast as export, accounting for actin nuclear accumulation. The faster shuttling dynamics correlated with reduced cellular retention of actin, and our data implicate actin polymerization in the stress-dependent uptake of nuclear actin. Furthermore, DNA replication stress induced a nuclear shift in IQGAP1 and Rac1 with enhanced import dynamics. Proximity ligation assays revealed that IQGAP1 associates in the nucleus with actin and Rac1, and formation of these complexes increased after hydroxyurea treatment. We propose that the replication stress checkpoint triggers co-ordinated nuclear entry and trafficking of actin, and of factors that regulate actin polymerization.
authors.affiliation: Westmead Institute for Cancer Research, The University of Sydney, Westmead, Australia.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Johnson
authors.forename: Manisha
authors.initials: M
authors.lastname: Sharma
authors.forename: Myth T S
authors.initials: MT
authors.lastname: Mok
authors.forename: Beric R
authors.initials: BR
authors.lastname: Henderson
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism*; D004260:DNA Repair / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects*; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D047468:Immunoprecipitation; D011485:Protein Binding; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D013936:Thymidine / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*
chemical_list: D000199:Actins; C101203:IQ motif containing GTPase activating protein 1; D034741:RNA, Small Interfering; D020703:ras GTPase-Activating Proteins; D020830:rac1 GTP-Binding Protein; D013936:Thymidine; D006918:Hydroxyurea
keywords: FRAP; HU; IQ-domain GTPase-activating protein 1; IQGAP1; IR; N-WASp; Nuclear actin; Nuclear transport; ROI; Rac1; Ras-related C3 botulinum toxin substrate 1; UV; fluorescence recovery after photobleaching; hydroxyurea; ionizing radiation; neural-Wiskott–Aldrich syndrome protein; region of interest; ultraviolet