Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism

Abstract

The actin cytoskeleton assembles into diverse load-bearing networks including stress fibers, muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl-1 & Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force-sensitivity. Zyxin rapidly localizes via its LIM domains to failing stress fibers in cells, known as strain sites, to initiate stress fiber repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We observe that many, but not all, LIM domains from functionally diverse proteins localize to spontaneous or induced stress fiber strain sites in mammalian cells. Additionally, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to stress fiber strain sites in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. Sequence analysis and mutagenesis of the LIM domain region of zyxin indicate a requirement of tandem LIM domains, which contribute additively, for sensing stress fiber strain sites. In vitro, purified LIM domains from mammalian zyxin and fission yeast Pxl1 bind to mechanically stressed F-actin networks but do not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress. Cells are subject to a wide range of omnipresent mechanical stimuli, which play essential physiological roles. Epithelial tissue stretch modulates cell proliferation 1,2 , blood pressure regulates the contractility of endothelial cells within blood vessels 3,4 , and muscle contraction shapes connective tissue remodeling 5 . Such mechanotransduction pathways allow for the integration of mechanical cues with the biochemical and genetic circuitry of the cell. While much progress has been made to elucidate the importance of mechanical stimuli in cell physiology, the underlying force-sensing mechanisms and organizational logic of many mechanotransduction pathways are unknown.To respond to mechanical cues and dynamically modulate cell mechanics, the actin cytoskeleton exploits force-sensitive biochemistry to construct actin filament (F-actin)-based network assemblies. Focal adhesions, the adhesive organelles between cells and their external matrix, can change in composition and size under varied mechanical load 6 . At the molecular scale, these focal adhesion changes arise primarily from force-dependent modulation of constituent proteins 6-8 . The force-dependent association of the focal adhesion proteins, vinculin and talin, to actin filaments is sensitive to filament polarity 9,10 , providing a mechanism to guide local cytoskeletal architecture under load. Protrusive forces at the leading edge of migrating cells are generated by actin po...

Spatial proximity of proteins surrounding zyxin under force-bearing conditions

“…These results suggest that zyxin recruitment to the force-bearing actin fibers does not require α-actinins or AFAP1. This is consistent with recent observations that zyxin binds directly to strained actin filaments (Sun et al, 2020;Winkelman et al, 2020).…”

De novo identification of universal cell mechanics gene signatures

“…The expression of calponin 2, likewise, stabilizes actin filaments and is increased in cells with high cytoskeletal tension 87,88 . Finally, FHL2 is a transcriptional coactivator that is found, together with other LIM domain protein families such as zyxin and paxillin, to localize to actin filaments that are under stress [65][66][67] . When the cytoskeletal tension is low, FHL2 translocates to the nucleus, thus serving as a nuclear transducer of actomyosin contractility 65 .…”

“…The second identified target, four and a half LIM domains 2, FHL2, is a multifaceted LIM domain protein with many binding partners and a transcription factor activity64 . FHL2 has recently been shown to remain bound to actin filaments under high tension, and be shuttled to the nucleus under low cytoskeletal tension65,66 -a property conserved among many LIM domain-containing proteins66,67 . The third target, Insulin-like growth factor binding protein 7, IGFBP7, is a secreted protein implicated in a variety of cancers.…”

Terminal web and vesicle trafficking proteins mediate nematode single-cell tubulogenesis

“…Future studies examining the biochemistry of EXC-1 and EXC-9 activation during canal development will be invaluable in understanding tubular growth mediated by these two proteins and by intermediate filaments. Recent manuscripts on bioRxiv ( Sun et al, 2020 Preprint ; Winkelman et al, 2020 Preprint ; not yet peer reviewed) suggest that other LIM-domain proteins are capable of measuring tension in cytoskeletal actin filaments. The growing excretory canal could provide an example of where the LIM domain of CRIP is performing a similar function with the stretchable domains of intermediate filaments.…”