Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin's mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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RhoA mediates stress fiber strain site recognition by the LIM protein testin

10.1101/2021.01.21.427693 ◽

2021 ◽

Author(s):

Stefano Sala ◽

Patrick W. Oakes

Keyword(s):

Actin Cytoskeleton ◽

Focal Adhesions ◽

Local Strain ◽

Stress Fibers ◽

Lim Domain ◽

Lim Protein ◽

Lim Domain Protein ◽

Domain Protein ◽

Site Recognition ◽

In Cells

AbstractThe actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize strained actin while the N-terminal domains alone recognize unstrained actin. Phosphorylation and cancer related mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes WT testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin’s mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state.SummaryLIM domain proteins recognize local strain in the actin cytoskeleton. This work suggests that the conformational state of the LIM protein testin determines its ability to recognize strained stress fibers and reveals a role for RhoA in regulating testin’s mechanosensitivity.

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REST/NRSF-Interacting LIM Domain Protein, a Putative Nuclear Translocation Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.23.24.9025-9031.2003 ◽

2003 ◽

Vol 23 (24) ◽

pp. 9025-9031 ◽

Cited By ~ 27

Author(s):

Masahito Shimojo ◽

Louis B. Hersh

Keyword(s):

Nuclear Localization ◽

Nuclear Translocation ◽

Protein A ◽

Lim Domain ◽

Nuclear Targeting ◽

Lim Domains ◽

Lim Domain Protein ◽

Domain Protein ◽

Nuclear Localization Sequences ◽

Novel Protein

ABSTRACT The transcriptional repressor REST/NRSF (RE-1 silencing transcription factor/neuron-restrictive silencer factor) and the transcriptional regulator REST4 share an N-terminal zinc finger domain structure involved in nuclear targeting. Using this domain as bait in a yeast two-hybrid screen, a novel protein that contains three LIM domains, putative nuclear localization sequences, protein kinase A phosphorylation sites, and a CAAX prenylation motif was isolated. This protein, which is localized around the nucleus, is involved in determining the nuclear localization of REST4 and REST/NRSF. We propose the name RILP, for REST/NRSF-interacting LIM domain protein, to label this novel protein. RILP appears to serve as a nuclear receptor for REST/NRSF, REST4, and possibly other transcription factors.

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

10.1101/2020.03.06.980649 ◽

2020 ◽

Author(s):

Jonathan D. Winkelman ◽

Caitlin A. Anderson ◽

Cristian Suarez ◽

David R. Kovar ◽

Margaret L. Gardel

Keyword(s):

Actin Cytoskeleton ◽

Fission Yeast ◽

Actin Filaments ◽

Mammalian Cells ◽

Stress Fiber ◽

Stress Fibers ◽

Strain Sensing ◽

Lim Domain ◽

Lim Domains ◽

Functionally Diverse

SUMMARYThe 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.

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Myofibril diameter is set by a finely tuned mechanism of protein oligomerization in Drosophila

eLife ◽

10.7554/elife.50496 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 7

Author(s):

Nicanor González-Morales ◽

Yu Shu Xiao ◽

Matthew Aaron Schilling ◽

Océane Marescal ◽

Kuo An Liao ◽

...

Keyword(s):

Muscle Function ◽

Developmental Stages ◽

Protein Oligomerization ◽

Lim Domain ◽

Muscle Type ◽

Lim Domains ◽

Lim Domain Protein ◽

Physiological Properties ◽

Domain Protein ◽

Underlying Mechanisms

Myofibrils are huge cytoskeletal assemblies embedded in the cytosol of muscle cells. They consist of arrays of sarcomeres, the smallest contractile unit of muscles. Within a muscle type, myofibril diameter is highly invariant and contributes to its physiological properties, yet little is known about the underlying mechanisms setting myofibril diameter. Here we show that the PDZ and LIM domain protein Zasp, a structural component of Z-discs, mediates Z-disc and thereby myofibril growth through protein oligomerization. Oligomerization is induced by an interaction of its ZM domain with LIM domains. Oligomerization is terminated upon upregulation of shorter Zasp isoforms which lack LIM domains at later developmental stages. The balance between these two isoforms, which we call growing and blocking isoforms sets the stereotyped diameter of myofibrils. If blocking isoforms dominate, myofibrils become smaller. If growing isoforms dominate, myofibrils and Z-discs enlarge, eventually resulting in large pathological aggregates that disrupt muscle function.

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PINCH2 is a new five LIM domain protein, homologous to PINCHand localized to focal adhesions☆

Experimental Cell Research ◽

10.1016/s0014-4827(02)00039-3 ◽

2003 ◽

Vol 284 (2) ◽

pp. 237-248 ◽

Cited By ~ 35

Author(s):

Attila Braun ◽

Randi Bordoy ◽

Fabio Stanchi ◽

Markus Moser ◽

G.ünter Kostka ◽

...

Keyword(s):

Focal Adhesions ◽

Lim Domain ◽

Lim Domain Protein ◽

Domain Protein

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Proteomic identification of the LIM domain protein FHL1 as the gene-product mutated in reducing body myopathy

Klinische Neurophysiologie ◽

10.1055/s-0029-1216187 ◽

2009 ◽

Vol 40 (01) ◽

Author(s):

J Schessl ◽

Y Zou ◽

MJ McGrath ◽

BS Cowling ◽

B Maiti ◽

...

Keyword(s):

Gene Product ◽

Lim Domain ◽

Lim Domain Protein ◽

Proteomic Identification ◽

Domain Protein

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Four-and-a-half LIM domain protein 2 (FHL2) deficiency protects mice from diet-induced obesity and high FHL2 expression marks human obesity

Metabolism ◽

10.1016/j.metabol.2021.154815 ◽

2021 ◽

pp. 154815

Author(s):

Maria P. Clemente-Olivo ◽

Jayron J. Habibe ◽

Mariska Vos ◽

Roelof Ottenhoff ◽

Aldo Jongejan ◽

...

Keyword(s):

Lim Domain ◽

Human Obesity ◽

Diet Induced Obesity ◽

Lim Domain Protein ◽

Domain Protein

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Four-and-a-half LIM-domain protein affects diet induced (hepatic) steatosis and lipid droplet formation

Journal of Hepatology ◽

10.1016/s0168-8278(18)30918-8 ◽

2018 ◽

Vol 68 ◽

pp. S348

Author(s):

J. Sommer ◽

C. Hellerbrand

Keyword(s):

Hepatic Steatosis ◽

Lipid Droplet ◽

Droplet Formation ◽

Lim Domain ◽

Lipid Droplet Formation ◽

Lim Domain Protein ◽

Domain Protein

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The LIM domain protein nTRIP6 modulates the dynamics of myogenic differentiation

Scientific Reports ◽

10.1038/s41598-021-92331-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tannaz Norizadeh Abbariki ◽

Zita Gonda ◽

Denise Kemler ◽

Pavel Urbanek ◽

Tabea Wagner ◽

...

Keyword(s):

Skeletal Muscle ◽

Satellite Cells ◽

Muscle Regeneration ◽

Myogenic Differentiation ◽

Skeletal Muscle Regeneration ◽

Temporal Control ◽

Lim Domain ◽

Lim Domain Protein ◽

Domain Protein

AbstractThe process of myogenesis which operates during skeletal muscle regeneration involves the activation of muscle stem cells, the so-called satellite cells. These then give rise to proliferating progenitors, the myoblasts which subsequently exit the cell cycle and differentiate into committed precursors, the myocytes. Ultimately, the fusion of myocytes leads to myofiber formation. Here we reveal a role for the transcriptional co-regulator nTRIP6, the nuclear isoform of the LIM-domain protein TRIP6, in the temporal control of myogenesis. In an in vitro model of myogenesis, the expression of nTRIP6 is transiently up-regulated at the transition between proliferation and differentiation, whereas that of the cytosolic isoform TRIP6 is not altered. Selectively blocking nTRIP6 function results in accelerated early differentiation followed by deregulated late differentiation and fusion. Thus, the transient increase in nTRIP6 expression appears to prevent premature differentiation. Accordingly, knocking out the Trip6 gene in satellite cells leads to deregulated skeletal muscle regeneration dynamics in the mouse. Thus, dynamic changes in nTRIP6 expression contributes to the temporal control of myogenesis.

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