Actin‐Based Motility Assay

Clainche, Christophe Le; Carlier, Marie-France

doi:10.1002/0471143030.cb1207s24

Cited by 18 publications

(15 citation statements)

References 21 publications

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“…Recombinant mouse IRSp53 I-BAR domain and ABBA I-BAR domain were purified and labeled as previously described ( Prévost et al, 2015 ) ( Saarikangas et al, 2009 ). Gelsolin was purified as previously described ( Le Clainche and Carlier, 2004 ).…”

Section: Methodsmentioning

confidence: 99%

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner

Tsai

Bertin

Bousquet

et al. 2018

eLife

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One challenge in cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat, positively or negatively curved. Using in vitro and cell biology approaches, we assess mechanisms of ezrin’s enrichment on curved membranes. We evidence that wild-type ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTD’s specific conformation reduces intermolecular interactions, allows binding to actin filaments, which reduces membrane tethering, and promotes ezrin binding to positively-curved membranes. While neither ezrinTD nor ezrinWT senses negative curvature alone, we demonstrate that interacting with curvature-sensing I-BAR-domain proteins facilitates ezrin enrichment in negatively-curved membrane protrusions. Overall, our work demonstrates that ezrin can tether membranes, or be targeted to curved membranes, depending on conformations and interactions with actin and curvature-sensing binding partners.

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Section: Methodsmentioning

confidence: 99%

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner

Tsai

Bertin

Bousquet

et al. 2018

eLife

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“…25), full-length human gelsolin, VCA domain of human neural Wiskott-Aldrich syndrome protein (N-WASP), the Arp2/3 complex from bovine brain or Spectrin-actin seeds from human erythrocytes were purified as described previously25405152.…”

Section: Methodsmentioning

confidence: 99%

Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

et al. 2016

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The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins.

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“…Full-length human gelsolin (29) was expressed and purified with modifications to previous methods as discussed in SI Materials and Methods (30). For the loaded in vitro motility assays, α-actinin was purified from chicken gizzards as previously described with a few minor changes (31).…”

Section: Methodsmentioning

confidence: 99%

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function

Sommese

Sung

Nag³

et al. 2013

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

156

229

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Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

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Actin‐Based Motility Assay

Cited by 18 publications

References 21 publications

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner

Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function

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