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Actin filaments as dynamic reservoirs for Drp1 recruitment.

ABSTRACT: Drp1 is a dynamin-family GTPase recruited to mitochondria and peroxisomes, where it oligomerizes and drives membrane fission. Regulation of mitochondrial Drp1 recruitment is not fully understood. We previously showed that Drp1 binds actin filaments directly, and actin polymerization is necessary for mitochondrial Drp1 oligomerization in mammals. Here we show the Drp1/actin interaction displays unusual properties that are influenced by several factors. At saturation, only a fraction Drp1 binds actin filaments, and the off-rate of actin-bound Drp1 is significantly increased by unbound Drp1. GDP and GTP accelerate and decelerate Drp1/actin binding dynamics, respectively. Actin has a biphasic effect on Drp1 GTP hydrolysis, increasing at low actin:Drp1 ratio but returning to baseline at high ratio. Drp1 also bundles filaments. Bundles have reduced dynamics but follow the same trends as single filaments. Drp1 preferentially incorporates into bundles at higher ionic strength. We measure Drp1 concentration to be ?0.5 ?M in U2OS cell cytosol, suggesting the actin-binding affinity measured here (Kd = 0.6 ?M) is in the physiologically relevant range. The ability of Drp1 to bind actin filaments in a highly dynamic manner provides potential for actin filaments to serve as reservoirs of oligomerization-competent Drp1 that can be accessed for mitochondrial fission.

SUBMITTER: Hatch AL

PROVIDER: S-EPMC5063618 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Actin filaments as dynamic reservoirs for Drp1 recruitment.

Hatch Anna L AL Ji Wei-Ke WK Merrill Ronald A RA Strack Stefan S Higgs Henry N HN

Molecular biology of the cell 20160824 20

Drp1 is a dynamin-family GTPase recruited to mitochondria and peroxisomes, where it oligomerizes and drives membrane fission. Regulation of mitochondrial Drp1 recruitment is not fully understood. We previously showed that Drp1 binds actin filaments directly, and actin polymerization is necessary for mitochondrial Drp1 oligomerization in mammals. Here we show the Drp1/actin interaction displays unusual properties that are influenced by several factors. At saturation, only a fraction Drp1 binds ac ...[more]

PMID: 27559132

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Project description:Experimental approaches such as fiber diffraction and cryo-electron microscopy reconstruction have defined regulatory positions of tropomyosin on actin but have not, as yet, succeeded at determining key atomic-level contacts between these proteins or fully substantiated the dynamics of their interactions at a structural level. To overcome this deficiency, we have previously employed computational approaches to deduce global dynamics of thin filament components by energy landscape determination and molecular dynamics simulations. Still, these approaches remain computationally challenging for any complex and large macromolecular assembly like the thin filament. For example, tropomyosin cable wrapping around actin of thin filaments features both head-to-tail polymeric interactions and local twisting, both of which depart from strict superhelical symmetry. This produces a complex energy surface that is difficult to model and thus to evaluate globally. Therefore, at this stage of our understanding, assessing global molecular dynamics can prove to be inherently impractical. As an alternative, we adopted a "divide and conquer" protocol to investigate actin-tropomyosin interactions at an atomistic level. Here, we first employed unbiased protein-protein docking tools to identify binding specificity of individual tropomyosin pseudorepeat segments over the actin surface. Accordingly, tropomyosin "ligand" segments were rotated and translated over potential "target" binding sites on F-actin where the corresponding interaction energetics of billions of conformational poses were ranked by the programs PIPER and ClusPro. These data were used to assess favorable interactions and then to rebuild models of seamless and continuous tropomyosin cables over the F-actin substrate, which were optimized further by flexible fitting routines and molecular dynamics. The models generated azimuthally distinct regulatory positions for tropomyosin cables along thin filaments on actin dominated by stereo-specific head-to-tail overlap linkage. The outcomes are in good agreement with current cryo-electron microscopy topology and consistent with long-thought residue-to-residue interactions between actin and tropomyosin.

Dataset Information

Actin filaments as dynamic reservoirs for Drp1 recruitment.

Publications

Actin filaments as dynamic reservoirs for Drp1 recruitment.

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