In vivo dynamics of the F-actin-binding protein neurabin-II.
Neurabin-II (spinophilin) is a ubiquitously expressed F-actin-binding protein 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 structure. We have stably expressed a green fluorescent protein (GFP)-tagged version 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-neurabin-II labels only a subset of F-actin within these cells, as indicated by rhodamine-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 actin 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 removal, recovery from which is greatly accelerated by stimulation of cells with epidermal growth factor (EGF). Furthermore, we show that this EGF-induced relocalization of GFP-neurabin-II is dependent on the activity of the small GTPase Rac1 but not the activity of ADP-ribosylation factor 6.
Stephens
D J
DJ
Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, U.K.
Banting
G
G
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002451:Cell Compartmentation / Q000187:drug effects; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D000099273:Neurabins
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D015638:Cytochalasin D; D004815:Epidermal Growth Factor; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D053778:Thiazolidines; D020830:rac1 GTP-Binding Protein; D000099273:Neurabins; C000715349:Arf6 protein, rat; C037068:latrunculin B