Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Stephens DJ

PROVIDER: S-EPMC1220745 | biostudies-literature | 2000 Jan

REPOSITORIES: biostudies-literature

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

Stephens D J DJ Banting G G

The Biochemical journal 20000101

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 ...[more]

PMID: 10620493

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Project description:We have used pulsed electron paramagnetic resonance, calorimetry, and molecular dynamics simulations to examine the structural mechanism of binding for dystrophin's N-terminal actin-binding domain (ABD1) and compare it to utrophin's ABD1. Like other members of the spectrin superfamily, dystrophin's ABD1 consists of two calponin-homology (CH) domains, CH1 and CH2. Several mutations within dystrophin's ABD1 are associated with the development of severe degenerative muscle disorders Duchenne and Becker muscular dystrophies, highlighting the importance of understanding its structural biology. To investigate structural changes within dystrophin ABD1 upon binding to actin, we labeled the protein with spin probes and measured changes in inter-CH domain distance using double-electron electron resonance. Previous studies on the homologous protein utrophin showed that actin binding induces a complete structural opening of the CH domains, resulting in a highly ordered ABD1-actin complex. In this study, double-electron electron resonance shows that dystrophin ABD1 also undergoes a conformational opening upon binding F-actin, but this change is less complete and significantly more structurally disordered than observed for utrophin. Using molecular dynamics simulations, we identified a hinge in the linker region between the two CH domains that grants conformational flexibility to ABD1. The conformational dynamics of both dystrophin's and utrophin's ABD1 showed that compact conformations driven by hydrophobic interactions are preferred and that extended conformations are energetically accessible through a flat free-energy surface. Considering that the binding free energy of ABD1 to actin is on the order of 6-7 kcal/mole, our data are compatible with a mechanism in which binding to actin is largely dictated by specific interactions with CH1, but fine tuning of the binding affinity is achieved by the overlap between conformational ensembles of ABD1 free and bound to actin.

Dataset Information

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

Publications

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

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