286, 26743C26753 [PMC free article] [PubMed] [Google Scholar] 22

286, 26743C26753 [PMC free article] [PubMed] [Google Scholar] 22. distributing or integrin activation by fibronectin to nuclear actin polymerization. Spreading-induced nuclear actin polymerization results in serum response factor (SRF)-mediated transcription through nuclear retention of myocardin-related transcription factor A (MRTF-A). Our results reveal a signaling pathway, which links integrin activation by extracellular matrix conversation to nuclear actin polymerization through the LINC complex, and therefore suggest a role for nuclear actin polymerization in the context of cellular adhesion and mechanosensing. represent S.E.; ****, 0.001. Data were collected from three impartial experiments; treated cells were compared with their respective control using a two-sided, unpaired Student’s test. observe Fig. 1and and Fig. 4, Febuxostat (TEI-6720) and symbolize S.E., ****, 0.001; represent S.E. 0.05. RESULTS AND Conversation We previously generated a nuclear actin probe by fusing LifeAct to an NLS (10). Although this allowed us to reliably detect endogenous nuclear actin polymerization and depolymerization, LifeAct can have some limitations as its expression level needs to be cautiously titrated and Febuxostat (TEI-6720) Aspn monitored to prevent any potential stabilization of put together F-actin structures. We therefore turned to a recently explained, antibody-based approach to visualize endogenous proteins utilizing the Chromobody technology (15). We targeted the Actin-Chromobody-TagGFP to the nucleus, generating Actin-Chromobody-TagGFP-NLS, herein termed nAC (Fig. 1and F-actin marker (Fig. 2non-spreading (control) cells. LifeAct-mCherry (to visualize cytoskeletal actin. and mark the corresponding nuclei of individual cells over time as they move through the visual field. Maximum intensity projections are shown. actin polymerization, we transfected NIH3T3-nAC cells with the nuclear targeted, non-polymerizable form of actin NLS-Flag-R62D (19, 20). These cells did not form FN-induced nuclear F-actin (Fig. 4, and and and and and (Fig. 5and and em G /em ). This demonstrates that nuclear Febuxostat (TEI-6720) formin activity is responsible for MRTF-A localization to the nucleus during cell distributing. Nuclear MRTF-A appearance and SRF activity appear to be slightly delayed in comparison with nuclear F-actin detection during distributing, possibly reflecting the notion that MRTF-A/SRF regulation is a consequence of nuclear actin assembly. Here we recognized an adhesion-triggered pathway that promotes the formation of nuclear F-actin during cell distributing (Fig. 6). Interestingly, although the shape of these nuclear filaments differs amazingly from those observed after serum activation (10), they appear to be nucleated by the same group of mDia formin regulators. Thus, different pathways may converge at nuclear formin activity to induce linear actin filaments of various length and business, further suggesting that additional yet unknown actin regulators cooperate. This is consistent with the view that many actin-regulating proteins are detectable in the nucleus (7, 28, 29). We find that spreading-induced nuclear actin assembly can regulate MRTF-A similar to the serum-induced response. However, the distributing response is much slower and more persistent in nature than the very rapid network formation, which occurs within seconds upon serum activation (Fig. 1 em C /em ). Thus, it seems tempting to speculate that additional nuclear functions may be regulated as a consequence of actin polymerization in the nucleus; spreading-mediated nuclear actin dynamics could be involved in changes in chromatin business (5, 30) or in the control of nuclear shape and positioning such as reported during cell migration (31, 32). Open in a separate window Physique 6. Graphic illustrating the current working model on nuclear F-actin formation induced by mechanotransduction via the LINC complex. Supplementary Material Supplemental Data: Click here to view. Acknowledgments We thank laboratory users for discussions. *This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (GR 2111/7-1). This short article contains supplemental Movies S1CS5. ?This short article was selected as a Paper of the Week. 2The abbreviations used are: LINClinker Febuxostat (TEI-6720) of nucleoskeleton and cytoskeletonMRTF-Amyocardin-related transcription factor ASRFserum response factorFNfibronectinnACnuclear Actin-ChromobodyNLSnuclear localization signalNESnuclear export signalTagGFPgreen fluorescent protein. Recommendations 1. Gama-Carvalho M., Carmo-Fonseca M. (2001) The rules and functions of nucleocytoplasmic shuttling proteins. FEBS Lett. 498, 157C163 [PubMed] [Google Scholar] 2. Floch A. G., Palancade B., Doye V. (2014) Fifty years of nuclear pores and nucleocytoplasmic transport studies: multiple tools revealing complex rules. Methods Cell Biol. 122, 1C40 [PubMed] [Google Scholar] 3. Dopie J., Skarp K. P., Rajakyl? E. K., Tanhuanp?? K., Vartiainen M. K. (2012) Active maintenance of nuclear actin by importin 9 supports transcription. Proc. Natl. Acad. Sci. U.S.A. 109, E544CE552 [PMC free article] [PubMed] [Google Scholar] 4. Stven T., Hartmann E., G?rlich D. (2003) Exportin 6: a novel nuclear export receptor that is specific for profilinactin complexes. EMBO J. 22, 5928C5940 [PMC free article] [PubMed] [Google Scholar] 5. Grosse R., Vartiainen M. K. (2013) To be or not to be put together: progressing into nuclear actin filaments. Nat. Rev. Mol. Cell Biol. 14, 693C697 [PubMed] [Google Scholar] 6. Treisman R. (2013) Shedding light on nuclear actin dynamics and function..