Filming Biomolecular Processes by High-Speed Atomic Force Microscopy

Ando, Toshio; Uchihashi, Takayuki; Scheuring, Simon

doi:10.1021/cr4003837

Cited by 315 publications

(297 citation statements)

References 387 publications

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“…For AFM imaging, the free oscillation amplitude was B2 nm and the set-point amplitude was B90% of the free oscillation amplitude. On the basis of this imaging condition, the tapping force estimated was less than 30 pN 31 . An amorphous carbon tip with the length of B1 mm was grown on the original tip by electron beam deposition and then the tip apex was sharpened by plasma etching under argon gas (B4 nm in radius).…”

Section: Methodsmentioning

confidence: 84%

Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

et al. 2014

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Processivity refers to the ability of synthesizing, modifying and degrading enzymes to catalyse multiple successive cycles of reaction with polymeric substrates without disengaging from the substrates. Since biomass polysaccharides, such as chitin and cellulose, often form recalcitrant crystalline regions, their degradation is highly dependent on the processivity of degrading enzymes. Here we employ high-speed atomic force microscopy to directly visualize the movement of two processive glycoside hydrolase family 18 chitinases (ChiA and ChiB) from the chitinolytic bacterium Serratia marcescens on crystalline b-chitin. The half-life of processive movement and the velocity of ChiA are larger than those of ChiB, suggesting that asymmetric subsite architecture determines both the direction and the magnitude of processive degradation of crystalline polysaccharides. The directions of processive movements of ChiA and ChiB are observed to be opposite. The molecular mechanism of the two-way traffic is discussed, including a comparison with the processive cellobiohydrolases of the cellulolytic system.

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Section: Methodsmentioning

confidence: 84%

Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

et al. 2014

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“…Although torsion and compaction are not exclusive, because both mechanisms could occur at the same time in constriction, we ought to visualize the global conformational changes of single dynamincoated membrane tubules with molecular and subsecond resolutions to better understand how dynamin constricts: we adapted in vitro reconstitution assays for high-speed AFM (HS-AFM) (13), which has recently proven powerful for the study of membrane remodeling proteins on mica-supported bilayers (14). We found that the adhesion of the proteins to the mica could impair the dynamin helix conformational change.…”

Section: Resultsmentioning

confidence: 99%

“…The resolution of the images on DOPS tubules was, however, insufficient to visualize the details of the helical reorganization process at the single-protein level, most probably because DOPS tubules have a low rigidity limiting HS-AFM resolution (13,20). To improve HS-AFM contouring and thus the resolution of the images, we opted for the use of rigid lipid nanorods formed by the spontaneous assembly of galactocerebrosides (21,22).…”

Section: Resultsmentioning

confidence: 99%

Dynamic remodeling of the dynamin helix during membrane constriction

Colom

Redondo-Morata

Chiaruttini

et al. 2017

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

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Dynamin is a dimeric GTPase that assembles into a helix around the neck of endocytic buds. Upon GTP hydrolysis, dynamin breaks these necks, a reaction called membrane fission. Fission requires dynamin to first constrict the membrane. It is unclear, however, how dynamin helix constriction works. Here we undertake a direct high-speed atomic force microscopy imaging analysis to visualize the constriction of single dynamin-coated membrane tubules. We show GTPinduced dynamic rearrangements of the dynamin helix turns: the average distances between turns reduce with GTP hydrolysis. These distances vary, however, over time because helical turns were observed to transiently pair and dissociate. At fission sites, these cycles of association and dissociation were correlated with relative lateral displacement of the turns and constriction. Our findings show relative longitudinal and lateral displacements of helical turns related to constriction. Our work highlights the potential of high-speed atomic force microscopy for the observation of mechanochemical proteins onto membranes during action at almost molecular resolution.dynamin | endocytosis | GTPase | high-speed atomic force microscopy | membrane fission

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“…These studies have been well documented in recent review articles (Ando et al 2014;Ando 2017a), and therefore I do not repeat the discussion here. Rather, I focus on in situ HS-AFM imaging of proteins existing on the surface of high-order structures, such as live cells.…”

Section: In Situ Hs-afm Imagingmentioning

confidence: 86%

High-speed atomic force microscopy and its future prospects

2017

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Various techniques have been developed and used to investigate how proteins produce complex biological architectures and phenomena. Among these techniques, high-speed atomic force microscopy (HS-AFM) holds a unique position. It is only HS-AFM that allows the simultaneous assessment of structure and dynamics of single protein molecules in action. This new microscopy tool has been successfully applied to a variety of proteins, from motor proteins to membrane proteins, antibodies, enzymes, and even to intrinsically disordered proteins. And yet there still remain many biomolecular phenomena that cannot be addressed by HS-AFM in its current form. Here, I present a brief history of HS-AFM development, describe the current state of HS-AFM, and then discuss which new biological scanning probe microscopy techniques will be coming up next.

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Filming Biomolecular Processes by High-Speed Atomic Force Microscopy

Cited by 315 publications

References 387 publications

Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

Dynamic remodeling of the dynamin helix during membrane constriction

High-speed atomic force microscopy and its future prospects

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