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Myosin-dependent actin stabilization as revealed by single-molecule imaging of actin turnover.

ABSTRACT: How mechanical stress applied to the actin network modifies actin turnover has attracted considerable attention. Actomyosin exerts the major force on the actin network, which has been implicated in actin stability regulation. However, direct monitoring of immediate changes in F-actin stability on alteration of actomyosin contraction has not been achieved. Here we reexamine myosin regulation of actin stability by using single-molecule speckle analysis of actin. To avoid possible errors attributable to actin-binding probes, we employed DyLight-labeled actin that distributes identical to F-actin in lamellipodia. We performed time-resolved analysis of the effect of blebbistatin on actin turnover. Blebbistatin enhanced actin disassembly in lamellipodia of fish keratocytes and lamellar of Xenopus XTC cells at an early stage of the inhibition, indicating that actomyosin contraction stabilizes cellular F-actin. In addition, our data show a previously unrecognized relationship between the actin network-driving force and the actin turnover rates in lamellipodia. These findings point to the power of direct viewing of molecular behavior in elucidating force regulation of actin filament turnover.

SUBMITTER: Yamashiro S

PROVIDER: S-EPMC6232968 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Myosin-dependent actin stabilization as revealed by single-molecule imaging of actin turnover.

Yamashiro Sawako S Tanaka Soichiro S McMillen Laura M LM Taniguchi Daisuke D Vavylonis Dimitrios D Watanabe Naoki N

Molecular biology of the cell 20180530 16

How mechanical stress applied to the actin network modifies actin turnover has attracted considerable attention. Actomyosin exerts the major force on the actin network, which has been implicated in actin stability regulation. However, direct monitoring of immediate changes in F-actin stability on alteration of actomyosin contraction has not been achieved. Here we reexamine myosin regulation of actin stability by using single-molecule speckle analysis of actin. To avoid possible errors attributab ...[more]

PMID: 29847209

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Project description:Localization of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to dendritic spine synapses is determined in part by the actin cytoskeleton. We determined binding of GFP-tagged CaMKII to tag-RFP-labeled actin cytoskeleton within live cells using total internal reflection fluorescence microscopy and single-molecule tracking. Stepwise photobleaching showed that CaMKII formed oligomeric complexes. Photoactivation experiments demonstrated that diffusion out of the evanescent field determined the track lifetimes. Latrunculin treatment triggered a coupled loss of actin stress fibers and the colocalized, long-lived CaMKII tracks. The CaMKIIα (α) isoform, which was previously thought to lack F-actin interactions, also showed binding, but this was threefold weaker than that observed for CaMKIIβ (β). The βE' splice variant bound more weakly than α, showing that binding by β depends critically on the interdomain linker. The mutations βT287D and αT286D, which mimic autophosphorylation states, also abolished F-actin binding. Autophosphorylation triggers autonomous CaMKII activity, but does not impair GluN2B binding, another important synaptic protein interaction of CaMKII. The CaMKII inhibitor tatCN21 or CaMKII mutations that inhibit GluN2B association by blocking binding of ATP (βK43R and αK42M) or Ca(2+)/calmodulin (βA303R) had no effect on the interaction with F-actin. These results provide the first rationale for the reduced synaptic spine localization of the αT286D mutant, indicating that transient F-actin binding contributes to the synaptic localization of the CaMKIIα isoform. The track lifetime distributions had a stretched exponential form consistent with a heterogeneously diffusing population. This heterogeneity suggests that CaMKII adopts different F-actin binding modes, which is most easily rationalized by multiple subunit contacts between the CaMKII dodecamer and the F-actin cytoskeleton that stabilize the initial weak (micromolar) monovalent interaction.

Dataset Information

Myosin-dependent actin stabilization as revealed by single-molecule imaging of actin turnover.

Publications

Myosin-dependent actin stabilization as revealed by single-molecule imaging of actin turnover.

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