Spatial proximity of proteins surrounding zyxin under force-bearing conditions

Abstract

Sensing physical forces is a critical first-step in mechano-transduction of cells. Zyxin, a LIM domain-containing protein, is recruited to force-bearing actin filaments and is thought to repair and strengthen them. Yet, the precise force-induced protein interactions surrounding zyxin remain unclear. Using Biotin IDentification (BioID) analysis, we identified proximal proteins surrounding zyxin under normal and force-bearing conditions by label-free mass spectrometry analysis. Under force-bearing conditions, increased biotinylation of α-actinin 1, α-actinin 4 and AFAP1 were detected, and these proteins accumulated along force-bearing actin fibers independently from zyxin, albeit at a lower intensity than zyxin. VASP also accumulated along force-bearing actin fibers in a zyxin-dependent manner, but the biotinylation of VASP remained constant regardless of force, supporting the model of a free zyxin-VASP complex in the cytoplasm being co-recruited to tensed actin fibers. In addition, ARHGAP42, a RhoA GAP, was also identified as a proximal protein of zyxin, and co-localized with zyxin along contractile actin bundles. The over-expression of ARHGAP42 reduced the rate of small wound closure, a zyxin-dependent process. These results demonstrate that the application of proximal biotinylation can resolve the proximity and composition of protein complexes as a function of force, which had not been possible with traditional biochemical analysis. [Media: see text] [Media: see text]

Cellular force‐sensing through actin filaments

“…Zyxin's N-terminus contains one binding site for the dimeric actin crosslinker a-actinin [66,67] and four binding sites for the tetrameric actin polymerization factor Ena/VASP [68,69]. While direct binding interactions between these proteins have been observed biochemically, the precise mechanism for coordinating SF repair remains unknown [58,[66][67][68][69]. A recent study reported that VASP promotes actin assembly by forming multivalent clusters on actin filaments with lamellipodin, a partner which features tandem VASP-binding sites similar to zyxin's [70].…”

“…1B). Within the cytoplasm, proteomics studies have revealed many LIM proteins to be enriched in stress fibres and focal adhesions in the presence of myosin activity [9–11,58]. Nuclear localization of several superfamily members has also independently been reported, where they have been suggested to regulate gene expression as transcriptional co‐activators [59].…”

Optical Fluorescence Imaging of Native Proteins Using a Fluorescent Probe with a Cell-Membrane-Permeable Carboxyl Group

“…Biotinylation and biotin/streptavidin affinity techniques are essential biosensing tools [ 40 , 41 ] in proteomics [ 40 , 42 ]. Proximity-dependent biotin identification (BioID) is a powerful tool to identify novel protein–protein and proximity-based interactions in living cells [ 43 , 44 ].…”

VASP localization to lipid bilayers induces polymerization driven actin bundle formation

“…In these multiprotein complexes, there are several proteins present with FPPPP-motifs to recruit VASP ( Renfranz and Beckerle, 2002 ). Zyxin ( Hoffman et al., 2006 ; Cheah et al., 2021 ), palladin ( Gateva et al., 2014 ), and vinculin ( Reinhard et al., 1996 ) are all situated at the actin-regulating area in the focal adhesion complex and can localize VASP to the forming actin filaments at the adhesion sites ( Kanchanawong et al., 2010 ). Therefore VASP can in biological systems be recruited by multiple different proteins from the cytosol toward the membrane to exert its function on actin.…”