Electron Microscopic Visualization of the Filament Binding Mode of Actin-Binding Proteins

Ito, Toshiro; Hirayama, Tasuku; Taki, Masayasu; Iyoshi, Shohei; Dai, Shuheng; Takeda, Susumu; Kimura-Sakiyama, Chieko; Oda, Tatsuya; Yamamoto, Yukio; Maéda, Yuichiro; Narita, Akihiro

doi:10.1016/j.jmb.2011.01.054

Cited by 3 publications

(7 citation statements)

References 51 publications

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“…In the present study, we directly determined the binding site of spire on the actin filament by employing a new method that combines Au-NP labeling and image analysis of the actin filament. 17 In the history of actin research, the interaction modes of actin-binding proteins with the actin filament have been elucidated by a series of rigorous biochemical experiments. The elucidation could be confirmed by visualizing the binding site of actin-binding proteins, using our novel imaging technique.…”

Section: Discussionmentioning

confidence: 99%

“…2e). 17 The Au-NP binds to the hexa-histidine tag at the N-terminus of spire through a Ni-charged chelator, nitrilotriacetic acid (NTA), on the surface of the Au-NP.…”

Section: Labeling Spire With the Au-npsmentioning

confidence: 99%

“…The Au-NPs with NTA were prepared. 17 After the addition of 1 μl of 20 mM Ni(NO 3 ) 2 to 100 μl of the Au-NP solution (10 μM) for the formation of Ni-NTA, 100 μl of a 50 μM His-Nspir or His-KIND solution, in 10 mM Tris-HCl (pH 8.0) and 200 mM NaCl, was mixed with the Au-NP solution. After incubation for 10 min at 4°C, the mixture was subjected to gel-filtration chromatography (Superose 6; GE Healthcare).…”

Section: Au-nspir Accelerates Actin Polymerizationmentioning

confidence: 99%

“…2e), we determined the polarity of individual actin filaments on electron micrographs by performing the image analysis technique that we previously established 17,18 (Supplementary Fig. 2).…”

Section: Au-nspir Binds To the B-end Of The Actin Filamentmentioning

confidence: 99%

“…We visualized the locations of the N-terminal half of spire (Nspir) labeled with gold nanoparticles (Au-NPs), which were developed in our laboratory. 17 The spire fragment, which was the same length as that used in the previous study, 11 accelerated actin polymerization. The polarity of the actin filament bound to spire was determined by our image analysis procedures.…”

mentioning

confidence: 92%

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Human Spire Interacts with the Barbed End of the Actin Filament

Ito

Narita

Hirayama

et al. 2011

Journal of Molecular Biology

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Section: Discussionmentioning

confidence: 99%

“…2e). 17 The Au-NP binds to the hexa-histidine tag at the N-terminus of spire through a Ni-charged chelator, nitrilotriacetic acid (NTA), on the surface of the Au-NP.…”

Section: Labeling Spire With the Au-npsmentioning

confidence: 99%

Section: Au-nspir Accelerates Actin Polymerizationmentioning

confidence: 99%

Section: Au-nspir Binds To the B-end Of The Actin Filamentmentioning

confidence: 99%

mentioning

confidence: 92%

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Human Spire Interacts with the Barbed End of the Actin Filament

Ito

Narita

Hirayama

et al. 2011

Journal of Molecular Biology

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Direct Determination of Actin Polarity in the Cell

Narita

Mueller

Urbán

et al. 2012

Journal of Molecular Biology

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Actin filaments are polar structures that exhibit a fast growing plus end and a slow growing minus end. According to their organization in cells, in parallel or antiparallel arrays, they can serve, respectively, in protrusions or in contractions. The determination of actin filament polarity in subcellular compartments is therefore required to establish their local function. Myosin binding has previously been the sole method of polarity determination. Here, we report the first direct determination of actin filament polarity in the cell without myosin binding. Negatively stained cytoskeletons of lamellipodia were analyzed by adapting electron tomography and a single particle analysis for filamentous complexes. The results of the stained cytoskeletons confirmed that all actin filament ends facing the cell membrane were the barbed ends. In general, this approach should be applicable to the analysis of actin polarity in tomograms of the actin cytoskeleton.

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Direct observation of the actin filament by tip-scan atomic force microscopy

et al. 2016

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Actin filaments, the actin–myosin complex and the actin–tropomyosin complex were observed by a tip-scan atomic force microscope (AFM), which was recently developed by Olympus as the AFM part of a correlative microscope. This newly developed AFM uses cantilevers of similar size as stage-scan AFMs to improve substantially the spatial and temporal resolution. Such an approach has previously never been possible by a tip-scan system, in which a cantilever moves in the x, y and z directions. We evaluated the performance of this developed tip-scan AFM by observing the molecular structure of actin filaments and the actin–tropomyosin complex. In the image of the actin filament, the molecular interval of the actin subunits (∼5.5 nm) was clearly observed as stripes. From the shape of the stripes, the polarity of the actin filament was directly determined and the results were consistent with the polarity determined by myosin binding. In the image of the actin–tropomyosin complex, each tropomyosin molecule (∼2 nm in diameter) on the actin filament was directly observed without averaging images of different molecules. Each tropomyosin molecule on the actin filament has never been directly observed by AFM or electron microscopy. Thus, our developed tip-scan AFM offers significant potential in observing purified proteins and cellular structures at nanometer resolution. Current results represent an important step in the development of a new correlative microscope to observe nm-order structures at an acceptable frame rate (∼10 s/frame) by AFM at the position indicated by the fluorescent dye observed under a light microscope.

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Electron Microscopic Visualization of the Filament Binding Mode of Actin-Binding Proteins

Cited by 3 publications

References 51 publications

Human Spire Interacts with the Barbed End of the Actin Filament

Human Spire Interacts with the Barbed End of the Actin Filament

Direct Determination of Actin Polarity in the Cell

Direct observation of the actin filament by tip-scan atomic force microscopy

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