Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155-157

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nature Methods (Impact Factor: 32.07). 03/2008; 5(2):155-7. DOI: 10.1038/nmeth.1176
Source: PubMed


We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

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Available from: Suliana Manley, Jul 13, 2015
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    • "F-actin by limiting access of these regulators to F-actin, which is inhibited by autophosphorylation within the F-actin binding domain. molecules using single-particle tracking photoactivated localization microscopy (sptPALM) (Manley et al., 2008; Frost et al., 2010; Lu et al., 2014). The distribution of localized molecules of mEos3-CaMKIIb WT and All A again revealed a greater enrichment within spines compared to that of the All D mutant (Figure 3D). "
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    ABSTRACT: The structural modification of dendritic spines plays a critical role in synaptic plasticity. CaMKII is a pivotal molecule involved in this process through both kinase-dependent and independent structural functions, but the respective contributions of these two functions to the synaptic plasticity remain unclear. We demonstrate that the transient interplay between the kinase and structural functions of CaMKII during the induction of synaptic plasticity temporally gates the activity-dependent modification of the actin cytoskeleton. Inactive CaMKII binds F-actin, thereby limiting access of actin-regulating proteins to F-actin and stabilizing spine structure. CaMKII-activating stimuli trigger dissociation of CaMKII from F-actin through specific autophosphorylation reactions within the F-actin binding region and permits F-actin remodeling by regulatory proteins followed by reassociation and restabilization. Blocking the autophosphorylation impairs both functional and structural plasticity without affecting kinase activity. These results underpin the importance of the interplay between the kinase and structural functions of CaMKII in defining a time window permissive for synaptic plasticity. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Aug 2015 · Neuron
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    • "Recently, assays such as uPAINT [19] or sptPALM [20] have allowed the generation of hundreds of trajectories in a short amount of time and thereby opened the door to efficient and detailed mapping of interaction sites of membrane molecules with submembrane scaffolds and their functional characterization [5, 21–23]. "
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    ABSTRACT: Single particle tracking is a powerful tool to investigate the function of biological molecules by following their motion in space. However, the simultaneous tracking of two different species of molecules is still difficult to realize without compromising the length or density of trajectories, the localization accuracy or the simplicity of the assay. Here, we demonstrate a simple dual color single particle tracking assay using small, bright, high-affinity labeling via nanobodies of accessible targets with widely available instrumentation. We furthermore apply a ratiometric step-size analysis method to visualize differences in apparent membrane viscosity.
    Full-text · Article · Jun 2015
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    • "This strictly limited the number of events that could be measured. This problem was eventually circumvented by tracking many temporally separated subsets of photoswitchable FP-tagged molecules in live-cell PALM experiments (sptPALM) (Hess et al., 2007; Manley et al., 2008). The high density of trajectories so obtained supplies enough statistical information to reconstruct high-resolution diffusion maps and infer distinct modes of diffusion. "
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    ABSTRACT: Observation of molecular processes inside living cells is fundamental to a quantitative understanding of how biological systems function. Specifically, decoding the complex behavior of single molecules enables us to measure kinetics, transport, and self-assembly at this fundamental level that is often veiled in ensemble experiments. In the past decade, rapid developments in fluorescence microscopy, fluorescence correlation spectroscopy, and fluorescent labeling techniques have enabled new experiments to investigate the robustness and stochasticity of diverse molecular mechanisms with high spatiotemporal resolution. This review discusses the concepts and strategies of structural and functional imaging in living cells at the single-molecule level with minimal perturbations to the specimen. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · May 2015 · Molecular cell
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