STUDIO DELL'INTERAZIONE TRA LE PROTEINE CITOSCHELETRICHE 4.1R80 E 4.1R135 E LA PROTEINA ICLN: ASPETTI MOLECOLARI E FUNZIONALI.

ICln is a multifunctional protein that has been implicated in many cellular functions, from cell volume regulation to RNA metabolism. It interacts with many members or regulators of the cytoskeleton, like the 4.1R protein, actin, myosin light chain and the JBP1 protein. This suggests that the cytoskeleton rearrangement that follows an hypotonic challenge could be involved in the translocation of the ICln protein from the cytosol to the cell membrane and in the activation of the ICl,swell. In this work we tried to elucidate the role of ICln-4.1R interaction in the context of Regulatory Volume Decrease; we focused on the in vivo interaction between ICln and the protein 4.1R by the use of the FRET technique. We used two different 4.1R isoforms for the FRET experiments: an high molecular weight (HMW) isoform (4.1 long) and a low molecular weight (LMW) one (4.1 short, without the initial 209 aa box). The strongest FRET signal was measured between ICln and 4.1Rsh, and it was significally increased by an hypotonic shock. Moreover, when the two proteins are co-expressed and interact, it seems that the two proteins change each other localization showing a decreased nuclear and, for 4.1R, membrane localization. From a functional point of view, preliminary patch-clamp experiments suggest that the over-expression of the 4.1sh isoform in HEK cells leads to an increase of the ICl,swell (the anion current that allows RVD) activation, thus confirming a role for 4.1R in cell volume regulation.