Mechanosensing through Direct Binding of Tensed F-Actin by LIM Domains

Sun, Xiaoyu; Phua, Donovan Y.Z.; Axiotakis, Lucas; Smith, Mark A.; Blankman, Elizabeth; Gong, Rui; Cail, Robert C.; Reyes, Santiago Espinosa de los; Beckerle, Mary C.; Waterman, Clare M.; Alushin, Gregory M.

doi:10.1016/j.devcel.2020.09.022

Cited by 100 publications

(129 citation statements)

References 66 publications

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“…Rap1 and its GEF Dzy are required for proper anisotropic apical constriction and ventral furrowing, and these factors may regulate these junctional rearrangements ( Sawyer et al, 2009 ; Spahn et al, 2012 ). Ventral junctions also recruit the tension-sensitive protein Ajuba ( Winkelman et al, 2020 ; Sun et al, 2020 ; Rauskolb et al, 2019 ). These results indicate that ventral cells undergo a number of changes that might alter their response to ectopic Rho1 activation.…”

Section: Discussionmentioning

confidence: 99%

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos

Rich

Fehon

Glotzer

2020

eLife

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Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.

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Section: Discussionmentioning

confidence: 99%

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos

Rich

Fehon

Glotzer

2020

eLife

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“…Structural information of individual LIM domains revealed specific sites of interactions, as for example between the ankyrin repeats of ILK and the first LIM domain of Pinch 23 . Although the biochemical and structural nature of most of the proposed LIM domain-mediated interactions is not yet defined precisely, many proteins containing multiple LIM domains are recruited to FAs under mechanical tension 30 – 33 . It was therefore postulated that LIM domains could function as tension sensors 32 and proposed to act as localizers, targeting proteins to specific subcellular locations, such as tensioned or injured F-actin networks 22 , 32 , 34 , 35 .…”

Section: Introductionmentioning

confidence: 99%

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvβ3 integrin-positive focal adhesions

et al. 2021

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The LIM domain-dependent localization of the adapter protein paxillin to β3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of β3 integrin at a putative paxillin binding site (β3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to β3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of β3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the β3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of β3 integrin-FAs.

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“…Based on 1) the similarities between the actin cables imaged by FIB-SEM (i.e., viewed in 2D as actin patches, Fig. 3) and the synaptopodinpositive patches at the periphery of the podocyte cell body and the major processes observed by STORM [31]; and 2) the localization of myosin IIA high in the podocytes (i.e., in the cell body and major processes but not in foot processes [31]), we speculate that these actin bundles observed by FIB-SEM are contractile in nature, which generate tension necessary for maturation of podocyte adhesive complexes at the GBM, analogous to tension thresholds for adhesion of other cells [61][62][63]. iv) After injury, podocytes undergo dramatic shape changes as foot processes become effaced, and this coincides with extensive actin rearrangements and the appearance of ectopic, irregularly thickened basal actin assemblies in the effaced areas.…”

Section: Discussionmentioning

confidence: 91%

3D Visualization of the Podocyte Actin Network using Integrated Membrane Extraction, Electron Microscopy, and Deep Learning

Roth

Loitman

et al. 2021

Preprint

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Although actin stress fibers are abundant in cultured cells, little is known about these structures in vivo. In podocytes of the kidney glomerulus, much evidence suggests that mechanobiological mechanisms underlie injury, with changes to actin stress fiber structures potentially responsible for pathological changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. We therefore developed the first visualization technique capable of resolving the three-dimensional (3D) podocyte actin network with unprecedented detail in healthy and injured podocytes, and applied this technique to reveal the changes in the actin network that occur upon podocyte injury. Using isolated glomeruli from healthy mice as well as from three different mouse injury models (Cd2ap-/-, Lamb2-/- and the Col4a3-/- model of Alport syndrome), we applied our novel imaging technique that integrates membrane-extraction, focused ion bean scanning electron microscopy (FIB-SEM), and deep learning image segmentation. In healthy glomeruli, we observed actin cables that link the interdigitating podocyte foot processes to newly described actin structures located at the periphery of the cell body. The actin cables within the foot processes formed a continuous, mesh-like, electron dense sheet that incorporated the slit diaphragms required for kidney filtration. After injury, the actin network was markedly different, having lost its organization and presenting instead as a disorganized assemblage of actin condensates juxtaposed to the glomerular basement membrane. The new visualization method enabled us, for the first time, to observe the detailed 3D organization of actin networks in both healthy and injured podocytes. Shared features of actin condensations across all three injury models further suggested common mechanobiological pathways that govern changes to podocyte morphology after injury.

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Mechanosensing through Direct Binding of Tensed F-Actin by LIM Domains

Cited by 100 publications

References 66 publications

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos

Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvβ3 integrin-positive focal adhesions

3D Visualization of the Podocyte Actin Network using Integrated Membrane Extraction, Electron Microscopy, and Deep Learning

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