An investigation of the differential function of actinin-1 and actinin-4

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Nathamuni Suresh, Apoorva
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University College Cork
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Actinins are involved in actin cross-linking and belong to the spectrin family of proteins. They are highly conserved and exist as dimers within the cell. Of the four isoforms of actinin, Actinin-1 (ACTN1) and Actinin-4 (ACTN4) are the non- muscle isoforms sharing a high sequence identity of nearly 87%. They play a significant role in cellular processes including cell migration, proliferation and adhesion. Apart from this, overexpression of Actinin-4 is also closely associated with the invasive phenotype of many cancers and used as a cancer prognosis marker in many cases. However, the molecular function of Actinin-4 overexpression in cancer is not well understood. Furthermore, the sub-cellular localization of the two non-muscle isoforms also varies considerably as reported in previous studies. The most striking difference was the localization of Actinin-4 to the nucleus, where Actinin-4 reportedly acts to modulate transcription. Actinin-1 has not been detected in the nucleus to date. Though neither Actinin-1 nor Actinin-4 contain a consensus nuclear localization signal sequence (NLS), the hydrophobic spectrin repeats (helical protein motifs acting as interaction sites for structural and signaling proteins) are hypothesized to allow its nucleo-cytoplasmic shuttling. Actinin-4 was found to have a consensus nuclear export sequence (NES) which allows its nuclear export mediated by CRM1. Actinin-1, with high sequence similarity to Actinin-4, also contains such a putative motif in its sequence. These observations raise the question as to why Actinin-4, and not Actinin-1, is localized to the nucleus. A possible explanation for such distinguishing characteristics could be unique interacting partners of Actinin-1 and Actinin-4. Another reason could be a higher affinity of Actinin-1 for its cytoskeletal protein partners, as compared to Actinin-4, which sequesters all available Actinin-1. Also it could also be possible that the putative NES-dependent nuclear export of Actinin-1 is much more efficient than its import, such that there is no Actinin-1 population in the nucleus. This work employs an immunoprecipitation assay to identify differences in the interacting partners of Actinin-1 and Actinin-4 in HEK293T cells to select proteins. The results proved that PDLIM1 can act as a positive control, showing strong interaction with both Actinin-1 and Actinin-4. Otherwise no statistically significant differences were observed between Actinin-1 and Actinin-4, though Actinin-4 shows subtly stronger interaction with AKT1 and RAVER1 as compared to the untransfected control. To study localization, fluorescence and confocal microscopy was employed to assess fluorescently tagged actinins. Though no differences were observed in localization of Actinin-1 and Actinin-4, under the experimental conditions employed, this study establishes an efficient system to study localization of Actinin-1 and Actinin-4 simultaneously, previously unutilized. Understanding the underlying molecular mechanism of the difference between the non-muscle actinin isoforms could help identify potential drug targets for cancer therapy.
Actinin-1 , Actinin-4
Nathamuni Suresh, A. 2020. An investigation of the differential function of actinin-1 and actinin-4. MRes Thesis, University College Cork.