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See detailInvestigation of the Nuclear Function of Tes, an Actin-Binding LIM Protein and Potential Tumor Suppressor
Vaccaroli, Raffaella UL

Doctoral thesis (2017)

The nucleus and the actin cytoskeleton are two distinct components of the eukaryotic cell. While the actin cytoskeleton is a dynamic structure that confers a high degree of cellular mobility as well as a ... [more ▼]

The nucleus and the actin cytoskeleton are two distinct components of the eukaryotic cell. While the actin cytoskeleton is a dynamic structure that confers a high degree of cellular mobility as well as a connection to the extracellular environment, the nucleus contains the genome and is strongly associated with gene expression from the steps of signal transduction to the actual transcription and the resulting translation. Although both components were initially studied separately, the ever-growing knowledge has lately asserted that they are tightly connected. In line, multitude of actin binding proteins were found to have roles in the nucleus, in addition to their well described roles in the cytoplasm. This research work focuses on the actin cytoskeleton protein Tes and its role in the nucleus. Tes is considered as a tumor suppressor protein, since its expression is reduced in different types of cancer cell lines and primary tumors, while its re-expression inhibits various aspects of cancer progression such as invasiveness and metastasis. As a cytoskeletal protein, Tes has been associated with actin polymerization as well as with cell migration and cell spreading. Our results show that, in addition to its cytoplasmic localization, Tes is localized in the nucleus. Artificial size increase of Tes, using a tag based on multiple GFP proteins, allowed us to assign Tes nuclear import to an active mechanism. Through fluorescence microscopy analysis of different modular fragments of Tes, we demonstrated that its PET and LIM1/2 domains are required for its nuclear localization. We also identified a classical monopartite nuclear localization signal (NLS) harbored within its PET domain, required for Tes import in the nucleus. In addition to Tes nuclear import, we also examined its nuclear export. Using the drug Leptomycin B, we demonstrated that Tes nuclear export is active and dependent on the CRM1-export mechanism. By combining protein sequence analysis with point mutations, we identified and characterized the nuclear export signal (NES) at the N-terminus of Tes. Using a photoconvertible probe, we demonstrated the presence of a slower photoconverted fraction of Tes in the nucleus, compared to the more dynamic cytoplasmic fraction. This prompted us to investigate further the existence of potential nuclear partners of Tes. Quantitative mass spectrometry analysis provided evidence for a potential involvement of Tes in nuclear pathways mediating the remodeling of actin, thus suggesting a novel role for Tes in the reorganization of the nuclear actin cytoskeleton. Furthermore, the proteomics studies associated the nuclear localization of Tes with signaling pathways promoting inflammation and cellular death. These results raised the hypothesis that Tes might promote tumor suppression not only through its cytoplasmic functions, but also through these newly described pathways connected to its nuclear localization. Further investigation will be necessary to fully elucidate the function of Tes in the nuclear compartment. [less ▲]

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