A hybrid high-speed atomic force–optical microscope for visualizing single membrane proteins on eukaryotic cells

Colom, Adai; Casuso, Ignacio; Rico, Félix; Scheuring, Simon

doi:10.1038/ncomms3155

Cited by 64 publications

(59 citation statements)

References 29 publications

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“…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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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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“…The use of fluorescence-related microscopy can help us to discern the detailed subcellular structures (e.g., cytoskeletons, cellular nuclei, cytoplasm) during the AFM indenting; thus, it greatly complemented the indentation by AFM. In addition, researchers have successfully combined high-speed AFM with fluorescence microscopy in the recent researches [82][83][84]. Suzuki et al [82] combined high-speed AFM with an optical fluorescence microscope by developing a tip-scanning system, and the results (observing the dynamics of living cells) showed that this system can remarkably improve the time resolution.…”

Section: Challenge and Outlookmentioning

confidence: 99%

“…Fukuda et al [83] have combined high-speed AFM with total internal reflection fluorescence microscopy, and this system can perform simultaneous topographic and fluorescence imaging at the single molecule level. Colom et al [84] have developed a hybrid high-speed atomic forceoptical microscope for visualizing single-membrane proteins on eukaryotic cells, allowing high-speed AFM imaging about 1,000 times faster than conventional AFM/ optical microscopy setups. The combination of high-speed AFM with fluorescence microscopy considerably accelerates the data acquisition rates of AFM, which is of evident significance in AFM nanoindentation.…”

Section: Challenge and Outlookmentioning

confidence: 99%

“…In order to address these issues, researchers from different disciplines (e.g., physics, engineering, biology, information, and automation) need to work together. In recent years, researchers are continuing to enhance and expand the performance of AFM indentation technique, such as high-speed AFM [82][83][84], tomographic contact resonance AFM [88], and force curvebased AFM [25,89]. The further expanding application of AFM indentation in the life sciences will bring tremendous changes in biomechanics, clinical medicine, and drug development.…”

Section: Challenge and Outlookmentioning

confidence: 99%

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Research progress in quantifying the mechanical properties of single living cells using atomic force microscopy

Liu

et al. 2014

Chin. Sci. Bull.

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The advent of atomic force microscopy (AFM) provides a powerful tool for investigating the behaviors of single living cells under near physiological conditions. Besides acquiring the images of cellular ultra-microstructures with nanometer resolution, the most remarkable advances are achieved on the use of AFM indenting technique to quantify the mechanical properties of single living cells. By indenting single living cells with AFM tip, we can obtain the mechanical properties of cells and monitor their dynamic changes during the biological processes (e.g., after the stimulation of drugs). AFM indentation-based mechanical analysis of single cells provides a novel approach to characterize the behaviors of cells from the perspective of biomechanics, considerably complementing the traditional biological experimental methods. Now, AFM indentation technique has been widely used in the life sciences, yielding a large amount of novel information that is meaningful to our understanding of the underlying mechanisms that govern the cellular biological functions. Here, based on the authors' own researches on AFM measurement of cellular mechanical properties, the principle and method of AFM indentation technique was presented, the recent progress of measuring the cellular mechanical properties using AFM was summarized, and the challenges of AFM single-cell nanomechanical analysis were discussed.

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“…Such a combined system has already been developed. However, its purpose is not the simultaneous observation of HS-AFM/fluorescence images but the positioning of the cantilever tip on a region of interest within a large object (e.g., eukaryotic cells) visualized with fluorescence microscopy (Colom et al 2013;Shibata et al 2015). In this hybrid system, the sample stage is scanned, and therefore the fluorescence image oscillates during HS-AFM imaging.…”

Section: Hybrid Hs-afm/optical Microscopy Systemmentioning

confidence: 99%

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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A hybrid high-speed atomic force–optical microscope for visualizing single membrane proteins on eukaryotic cells

Cited by 64 publications

References 29 publications

Dynamic remodeling of the dynamin helix during membrane constriction

Dynamic remodeling of the dynamin helix during membrane constriction

Research progress in quantifying the mechanical properties of single living cells using atomic force microscopy

High-speed atomic force microscopy and its future prospects

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