Single-Molecule Imaging Techniques to Visualize Chemotactic Signaling Events on the Membrane of Living Dictyostelium Cells

Miyanaga, Yukihiro; Matsuoka, Satomi; Ueda, Masahiro

doi:10.1007/978-1-60761-198-1_28

Cited by 30 publications

(33 citation statements)

References 19 publications

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“…In Fig. 2B, the number of molecules that remained bound to the membrane was counted every 33 msec beginning from the onset of membrane association, normalized by the total number of molecules and plotted against time [23], [24]. The decay rate of the number of molecules on the membrane, which corresponds to the membrane dissociation rate, varied depending on the number of mutations.…”

Section: Resultsmentioning

confidence: 99%

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PTEN Hopping on the Cell Membrane Is Regulated via a Positively-Charged C2 Domain

2014

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PTEN, a tumor suppressor that is frequently mutated in a wide spectrum of cancers, exerts PI(3,4,5)P3 phosphatase activities that are regulated by its dynamic shuttling between the membrane and cytoplasm. Direct observation of PTEN in the interfacial environment can offer quantitative information about the shuttling dynamics, but remains elusive. Here we show that positively charged residues located in the cα2 helix of the C2 domain are necessary for the membrane localization of PTEN via stable electrostatic interactions in Dictyostelium discoideum. Single-molecule imaging analyses revealed that PTEN molecules moved distances much larger than expected had they been caused by lateral diffusion, a phenomenon we call “hopping.” Our novel single-particle tracking analysis method found that the cα2 helix aids in regulating the hopping and stable-binding states. The dynamically established membrane localization of PTEN was revealed to be essential for developmental processes and clarified a fundamental regulation mechanism of the protein quantity and activity on the plasma membrane.

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Section: Resultsmentioning

confidence: 99%

“…Single molecules of wild type PTEN-Halo and PTEN i -Halo were visualized through objective type TIRFM. Details of the TIRFM configuration are described elsewhere [23].…”

Section: Methodsmentioning

confidence: 99%

PTEN Hopping on the Cell Membrane Is Regulated via a Positively-Charged C2 Domain

2014

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“…Single-particle tracking (SPT) methods have been used to study the mobility of GPCR fusion proteins of with fluorescent proteins [63, 64], enzyme tags (HaloTag or ACP tag) that allow specific conjugation with fluorophores [65, 66], or epitope tags that allow visualization with gold-tagged Fab antibody fragments [63]. SPT has been used to infer receptor binding of fluorescent protein fusion constructs of heterotrimeric G proteins [67, 68].…”

Section: Escaping the Flatlands One Molecule At A Time: Concluding Rementioning

confidence: 99%

Escaping the flatlands: new approaches for studying the dynamic assembly and activation of GPCR signaling complexes

Huber¹,

Sakmar²

2011

Trends in Pharmacological Sciences

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Despite significant recent advances in molecular and structural studies of G protein-coupled receptors (GPCRs), understanding transmembrane signal transduction with chemical precision requires new approaches. Simple binary receptor-ligand or receptor-G protein complex models cannot describe adequately the relevant macromolecular signaling machines. GPCR “signalosomes” undergo complex dynamic assembly/disassembly reactions to create allosteric signaling conduits whose properties cannot necessarily be predicted from individual elements alone. The combinatorial possibilities inherent in a system with hundreds of potential components suggest that high-content miniaturized experimental platforms and computational approaches will be required. To study allosteric effects involved in signalosome reaction pathways, a bottom-up approach is proposed to use multicolor single-molecule detection fluorescence experiments in biochemically defined systems complemented by molecular dynamics models of macromolecular complexes. In bridging the gap between molecular and systems biology, this synthetic approach suggests a way forward from the flatlands to multi-dimensional data collection.

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“…Optical microscopic methods, such as fluorescent video imaging, show molecular dynamics in single living cells. 3,4 The optical microscopic technique has been applied to imaging of bacterial cells and provides insights about molecular dynamics in vivo. For example, Kim et al observed the dynamics of the single cytoskeletal protein MreB in living bacterial cells using a total internal reflection fluorescence microscope.…”

Section: Introductionmentioning

confidence: 99%

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Yamashita

Taoka

Uchihashi

et al. 2012

Journal of Molecular Biology

118

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Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells.In the present study, we used high-speed atomic force microscopy (HS-AFM) to image the molecular dynamics of living bacterial cell surfaces. HS-AFM visualizes the dynamic molecular processes of isolated proteins at sub-molecular resolution without the need for complicated sample preparation. In the present study, magnetotactic bacterial cells were anchored in liquid medium on substrate modified by poly-L-lysine and glutaraldehyde. High-resolution HS-AFM images of live cell surfaces showed that the bacterial outer membrane was covered with a net-like structure comprising holes and the hole rims framing them. Furthermore, HS-AFM captured the dynamic movement of the surface ultrastructure, showing that the holes in the net-like structure slowly diffused in the cell surface. Nano-dissection revealed that porin trimers constitute the net-like structure. Here, we report for the first time the direct observation of dynamic molecular architectures on a live cell surface using HS-AFM.

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Single-Molecule Imaging Techniques to Visualize Chemotactic Signaling Events on the Membrane of Living Dictyostelium Cells

Cited by 30 publications

References 19 publications

PTEN Hopping on the Cell Membrane Is Regulated via a Positively-Charged C2 Domain

PTEN Hopping on the Cell Membrane Is Regulated via a Positively-Charged C2 Domain

Escaping the flatlands: new approaches for studying the dynamic assembly and activation of GPCR signaling complexes

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

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