In vivo dynamics of the F-actin-binding protein neurabin-II

Neurabin-II (spinophilin) is a ubiquitously expressed F-actin-binding prote in containing an N-terminal actin-binding domain, a PDZ (PSD95/discs large/ ZO-1) domain and a C-terminal domain predicted to form a coiled-coil struct ure. We have stably expressed a green fluorescent protein (GFP)-tagged vers ion of neurabin-II in PC12 cells, and characterized the in vivo dynamics of this actin-binding protein using confocal fluorescence microscopy. We show that GFP-neurabin-II localizes to actin filaments, especially at cortical sites and areas underlying sites of active membrane remodelling. GFP-neurab in-II labels only a subset of F-actin within these cells, as indicated by r hodamine-phalloidin staining. Both actin filaments and small, highly motile structures within the cell body are seen. Photobleaching experiments show that GFP-neurabin-II also exhibits highly dynamic behaviour when bound to a ctin filaments. Latrunculin B treatment results in rapid relocalization of GFP-neurabin-II to the cytosol, whereas cytochalasin D treatment causes the collapse of GFP-neurabin-II fluorescence to intensely fluorescent foci of F-actin within the cell body. This collapse is reversed on cytochalasin D r emoval, recovery from which is greatly accelerated by stimulation of cells with epidermal growth factor (EGF). Furthermore, we show that this EGF-indu ced relocalization of GFP-neurabin-II is dependent on the activity of the s mall GTPase Rad but not the activity of ADP-ribosylation factor 6.