Bacher, C.P., Reichenzeller, M., Athale, C., Herrmann, H. & Eils, R. 4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin-poor tracks. BMC Cell. Biol. 5, 45

Division of Theoretical Bioinformatics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. <>
BMC Cell Biology (Impact Factor: 2.34). 12/2004; 5(1):45. DOI: 10.1186/1471-2121-5-45
Source: PubMed


The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei.
We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 mum - wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data.
Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

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Available from: Chaitanya Athale, Oct 09, 2015
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    • "The mean square displacement (MSD) was calculated for each individual particle and plotted versus Δt. FS's movements were classified into three groups of motility based on theoretical previous studies [29–31]: static (particle show little to no motion; MSD curve is a straight line and α ≤ 0.1 ), Diffusion (MSD increases linearly with the time intervals and 0.1< α < 1.2) or directed motion (MSD plot gives a parabolic curve and α ≥ 1.2) (Figure 5a, 5a’,5a’’) (movie S9). Interestingly, only 6% of injected FS-PEG-OMe show directed motion compared to approximately 22% for FS-PEG-WT, 23% FS-PEG-BS69 and 25% for FS-PEG-WTY-2 with average speeds of 0.4 μm/s, consistent with active transport (Figure 5b). "
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    ABSTRACT: Molecular motors transport various cargoes including vesicles, proteins and mRNAs, to distinct intracellular compartments. A significant challenge in the field of nanotechnology is to improve drug nuclear delivery by engineering nanocarriers transported by cytoskeletal motors. However, suitable in vivo models to assay transport and delivery efficiency remain very limited. Here, we develop a fast and genetically tractable assay to test the efficiency and dynamics of fluospheres (FS) using microinjection into Drosophila oocytes coupled with time-lapse microscopy. We designed dynein motor driven FS using a collection of dynein light chain 8 (LC8) peptide binding motifs as molecular linkers and characterized in real time the efficiency of the FS movement according to its linker's sequence. Results show that the conserved LC8 binding motif allows fast perinuclear nanoparticle's accumulation in a microtubule and dynein dependent mechanism. These data reveal the Drosophila oocyte as a new valuable tool for the design of motor driven nanovectors.
    PLoS ONE 12/2013; 8(12):e82908. DOI:10.1371/journal.pone.0082908 · 3.23 Impact Factor
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    • "(A) Simulated mean squared displacement plots for particle tracking. I shows diffusion for confined molecules, II shows obstructed diffusion, III normal diffusion, and IV shows MSD for directed motion; (B) shows example particle tracks for the motion types plotted in (A) [115]. "
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    ABSTRACT: Insights from single-molecule tracking in mammalian cells have the potential to greatly contribute to our understanding of the dynamic behavior of many protein families and networks which are key therapeutic targets of the pharmaceutical industry. This is particularly so at the plasma membrane, where the method has begun to elucidate the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell, including signal transduction, receptor recognition, cell-cell adhesion, etc. However, despite much progress, single-molecule tracking faces challenges in mammalian samples that hinder its general application in the biomedical sciences. Much work has recently focused on improving the methods for fluorescent tagging of target molecules, detection and localization of tagged molecules, which appear as diffraction-limited spots in charge-coupled device (CCD) images, and objectively establishing the correspondence between moving particles in a sequence of image frames to follow their diffusive behavior. In this review we outline the state-of-the-art in the field and discuss the advantages and limitations of the methods available in the context of specific applications, aiming at helping researchers unfamiliar with single molecules methods to plan out their experiments.
    International Journal of Molecular Sciences 12/2012; 13(11):14742-65. DOI:10.3390/ijms131114742 · 2.86 Impact Factor
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    • "For instance we may be interested in the trafficking of a set of fluorescently-labelled particles which are driven intermittantly by molecular motors on microtubules and F-actin, but these may also experience advection in the flowing cytoplasm, and Brownian motion. A simple way of analysing such complicated motion is to reduce its noise by spatial and temporal filtering, and break it up into different components [1] [2] [3] [5] [6] [8]. These procedures are useful and conceptually straight-forward, but have obvious disadvantages in that they all involve " fudge factors " , such as smoothing length scales and time scales, or thresholds to distinguish how fast, straight or long-lived a track must be in order to count as a segment of motion we want to measure. "
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    ABSTRACT: The first passage probability (FPP), of trafficked intracellular particles reaching a displacement L, in a given time t or inverse velocity S = t/L, can be calculated robustly from measured particle tracks. The FPP gives a measure of particle movement in which different types of motion, e.g. diffusion, ballistic motion, and transient run-rest motion, can readily be distinguished in a single graph, and compared with mathematical models. The FPP is attractive in that it offers a means of reducing the data in the measured tracks, without making assumptions about the mechanism of motion. For example, it does not employ smoothing, segmentation or arbitrary thresholds to discriminate between different types of motion in a particle track. In contrast to conventional mean square displacement analysis, FPP is sensitive to a small population of trafficked particles that move long distances (> or = 5 microm), which are thought to be crucial for efficient long range signaling in theories of network dynamics. Taking experimental data from tracked endocytic vesicles, and calculating the FPP, we see how molecular treatments affect the trafficking. We show the FPP can quantify complicated movement which is neither completely random nor completely deterministic, making it highly applicable to trafficked particles in cell biology.
    Physical Chemistry Chemical Physics 04/2010; 12(15):3753-61. DOI:10.1039/b921874b · 4.49 Impact Factor
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