Villidin, a Novel WD-repeat and Villin-related Protein fromDictyostelium,Is Associated with Membranes and the Cytoskeleton

Villidin is a novel multidomain protein (190 kDa) from Dictyostelium amoebae containing WD repeats at its N-terminus, three PH domains in the middle of the molecule, and five gelsolin-like segments at the C-terminus, followed by a villin-like headpiece. Villidin mRNA and protein are present in low amounts during growth and early aggregation, but increase during development and reach their highest levels at the tipped mound stage. The protein is present in the cytosol as well as in the cytoskeletal and membrane fractions. GFP-tagged full-length villidin exhibits a similar distribution as native villidin, including a distinct colocalization with Golgi structures. Interestingly, GFP fusions with the gelsolin/villin-like region are uniformly dispersed in the cytoplasm, whereas GFP fusions of the N-terminal WD repeats codistribute with F-actin and are associated with the Triton-insoluble cytoskeleton. Strains lacking villidin because of targeted deletion of its gene grow normally and can develop into fruiting bodies. However, cell motility is reduced during aggregation and phototaxis is impaired in the mutant strains. We conclude that villidin harbors a major F-actin binding site in the N-terminal domain and not in the villin-like region as expected; association of villidin with vesicular membranes suggests that the protein functions as a linker between membranes and the actin cytoskeleton.

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Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity

The Journal of Cell Biology ◽

10.1083/jcb.201510012 ◽

2016 ◽

Vol 213 (3) ◽

pp. 371-383 ◽

Cited By ~ 112

Author(s):

Abhishek Kumar ◽

Mingxing Ouyang ◽

Koen Van den Dries ◽

Ewan James McGhee ◽

Keiichiro Tanaka ◽

...

Keyword(s):

Adhesion Force ◽

Focal Adhesions ◽

Actin Binding ◽

Force Transmission ◽

C Terminus ◽

Tension Sensor ◽

Cellular Mechanosensing ◽

The Difference ◽

Development And Validation ◽

Actin Binding Site

Integrin-dependent adhesions are mechanosensitive structures in which talin mediates a linkage to actin filaments either directly or indirectly by recruiting vinculin. Here, we report the development and validation of a talin tension sensor. We find that talin in focal adhesions is under tension, which is higher in peripheral than central adhesions. Tension on talin is increased by vinculin and depends mainly on actin-binding site 2 (ABS2) within the middle of the rod domain, rather than ABS3 at the far C terminus. Unlike vinculin, talin is under lower tension on soft substrates. The difference between central and peripheral adhesions requires ABS3 but not vinculin or ABS2. However, differential stiffness sensing by talin requires ABS2 but not vinculin or ABS3. These results indicate that central versus peripheral adhesions must be organized and regulated differently, and that ABS2 and ABS3 have distinct functions in spatial variations and stiffness sensing. Overall, these results shed new light on talin function and constrain models for cellular mechanosensing.

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Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

Molecular Biology of the Cell ◽

10.1091/mbc.6.8.1061 ◽

1995 ◽

Vol 6 (8) ◽

pp. 1061-1075 ◽

Cited By ~ 290

Author(s):

R Gary ◽

A Bretscher

Keyword(s):

Amino Acids ◽

Binding Site ◽

Sodium Dodecyl ◽

Actin Binding ◽

Terminal Domain ◽

Cell Extracts ◽

C Terminus ◽

Self Association ◽

Actin Binding Site

Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.

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