Identification and characterization of multiple similar ligand-binding repeats in filamin.

Ithychanda, Sujay Subbayya; Hsu, Dennis J.; Li, Hanhan; Yan, Linda; Liu, David D.; Das, Mitali; Plow, Edward F.; Qin, Jun

doi:10.1074/jbc.a109.060954

Cited by 16 publications

(37 citation statements)

References 24 publications

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“…S4 A and B). Even though the binding kinetics of those ligands have not been measured before, the equilibrium binding constants (K D ) of the three peptides calculated from the kinetics agree well with bulk measurements (Table S3) (25). It is interesting to note that with K D values in the micromolar range and above, as well as lifetimes below a second, all those interactions are very dynamic.…”

Section: Resultssupporting

confidence: 61%

“…The subsequent build-up of mechanical tension can then relieve the autoinhibition of the other domains promoting strong anchoring. The necessity of multiple parallel bonds for stable anchoring is in accord with the idea that the multiple binding sites of filamin can also induce clustering of transmembrane receptors (10,25). Alternatively, filamin interactions may be substituted or stabilized by other adaptor molecules.…”

Section: Discussionsupporting

confidence: 54%

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Dynamic force sensing of filamin revealed in single-molecule experiments

Rognoni

Stigler

Pelz

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

149

160

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Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20-21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that filamin force sensing is a highly dynamic process occurring in rapid equilibrium that increases the affinity to the target peptides by up to a factor of 17 between 2 and 5 pN. The equilibrium mechanism we find here can offer a general scheme for cellular force sensing.optical tweezers | mechanosensing

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Section: Resultssupporting

confidence: 61%

Section: Discussionsupporting

confidence: 54%

Dynamic force sensing of filamin revealed in single-molecule experiments

Rognoni

Stigler

Pelz

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

149

160

View full text Add to dashboard Cite

show abstract

“…Sequence and structural analysis has revealed that FLN repeats exist as domain pairs e.g. 16/17, 18/19 and 20/21 (Lad et al 2007;Heikkinen et al 2009) with multiple β-integrin interaction sites functionally identified (Ithychanda et al 2009). …”

Section: Filamin Domain Organisation and Structurementioning

confidence: 99%

“…The issue of whether FLNs can heterodimerise is not fully resolved; FLNB/C repeat 24 heterodimers were observed by in vitro cross-linking assays, but the combinations of FLNA/B or FLNA/C were not (Himmel et al Fig. 1 Schematic representation of filamin cross-linking F-actin and clustering integrin receptors at the plasma membrane (Ithychanda et al 2009) (Sheen et al 2002).…”

Section: Filamin Domain Organisation and Structurementioning

confidence: 99%

“…When external force is applied to integrins, forces are transmitted via cytoskeletal filaments leading to physical rearrangements at the nucleus (Maniotis et al 1997) with upregulation of FLNA expression (D'Addario et al 2001). FLN shows differential affinities for different β-integrin isoforms (Pfaff et al 1998), and the location of multiple integrin binding sites along the rod domain suggests that FLN may be responsible for integrin clustering (Ithychanda et al 2009) leading to the inhibition of cell migration and polarisation via reduced membrane remodelling (Calderwood et al 2001).…”

Section: Filamin Cellular Functionsmentioning

confidence: 99%

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Filamin structure, function and mechanics: are altered filamin-mediated force responses associated with human disease?

Sutherland-Smith

2011

Biophys Rev

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The cytoskeleton framework is essential not only for cell structure and stability but also for dynamic processes such as cell migration, division and differentiation. The F-actin cytoskeleton is mechanically stabilised and regulated by various actin-binding proteins, one family of which are the filamins that cross-link F-actin into networks that greatly alter the elastic properties of the cytoskeleton. Filamins also interact with cell membraneassociated extracellular matrix receptors and intracellular signalling proteins providing a potential mechanism for cells to sense their external environment by linking these signalling systems. The stiffness of the external matrix to which cells are attached is an important environmental variable for cellular behaviour. In order for a cell to probe matrix stiffness, a mechanosensing mechanism functioning via alteration of protein structure and/or binding events in response to external tension is required. Current structural, mechanical, biochemical and human disease-associated evidence suggests filamins are good candidates for a role in mechanosensing.

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Novel interactors and a role for supervillin in early cytokinesis

2010

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Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, FLNA, HAX1, and MIF4GD also mimic supervillin over-expression by inhibiting cell spreading. Most new interactors have known roles in supervillin-associated processes, e.g. cell motility, membrane trafficking, ERK signaling, and matrix invasion; three (KIF14, KIFC3, STARD9/KIF16A) have kinesin motor domains; and five (EPLIN, KIF14, BUB1, ODF2/cenexin, RHAMM) are important for cell division. GST fusions of the supervillin G2–G3 or G4–G6 repeats co-sediment KIF14 and EPLIN, respectively, consistent with a direct association. Supervillin depletion leads to increased numbers of bi- and multi-nucleated cells. Cytokinesis failure occurs predominately during early cytokinesis. Supervillin localizes with endogenous myosin II and EPLIN in the cleavage furrow, and overlaps with the oncogenic kinesin, KIF14, at the midbody. We conclude that supervillin, like its interactors, is important for efficient cytokinesis. Our results also suggest that supervillin and its interaction partners coordinate actin and microtubule motor functions throughout the cell cycle.

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Identification and characterization of multiple similar ligand-binding repeats in filamin.

Cited by 16 publications

References 24 publications

Dynamic force sensing of filamin revealed in single-molecule experiments

Dynamic force sensing of filamin revealed in single-molecule experiments

Filamin structure, function and mechanics: are altered filamin-mediated force responses associated with human disease?

Novel interactors and a role for supervillin in early cytokinesis

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