6,457 research outputs found
Anomalous transport in the crowded world of biological cells
A ubiquitous observation in cell biology is that diffusion of macromolecules
and organelles is anomalous, and a description simply based on the conventional
diffusion equation with diffusion constants measured in dilute solution fails.
This is commonly attributed to macromolecular crowding in the interior of cells
and in cellular membranes, summarising their densely packed and heterogeneous
structures. The most familiar phenomenon is a power-law increase of the MSD,
but there are other manifestations like strongly reduced and time-dependent
diffusion coefficients, persistent correlations, non-gaussian distributions of
the displacements, heterogeneous diffusion, and immobile particles. After a
general introduction to the statistical description of slow, anomalous
transport, we summarise some widely used theoretical models: gaussian models
like FBM and Langevin equations for visco-elastic media, the CTRW model, and
the Lorentz model describing obstructed transport in a heterogeneous
environment. Emphasis is put on the spatio-temporal properties of the transport
in terms of 2-point correlation functions, dynamic scaling behaviour, and how
the models are distinguished by their propagators even for identical MSDs.
Then, we review the theory underlying common experimental techniques in the
presence of anomalous transport: single-particle tracking, FCS, and FRAP. We
report on the large body of recent experimental evidence for anomalous
transport in crowded biological media: in cyto- and nucleoplasm as well as in
cellular membranes, complemented by in vitro experiments where model systems
mimic physiological crowding conditions. Finally, computer simulations play an
important role in testing the theoretical models and corroborating the
experimental findings. The review is completed by a synthesis of the
theoretical and experimental progress identifying open questions for future
investigation.Comment: review article, to appear in Rep. Prog. Phy
Inferring diffusion in single live cells at the single molecule level
The movement of molecules inside living cells is a fundamental feature of
biological processes. The ability to both observe and analyse the details of
molecular diffusion in vivo at the single molecule and single cell level can
add significant insight into understanding molecular architectures of diffusing
molecules and the nanoscale environment in which the molecules diffuse. The
tool of choice for monitoring dynamic molecular localization in live cells is
fluorescence microscopy, especially so combining total internal reflection
fluorescence (TIRF) with the use of fluorescent protein (FP) reporters in
offering exceptional imaging contrast for dynamic processes in the cell
membrane under relatively physiological conditions compared to competing single
molecule techniques. There exist several different complex modes of diffusion,
and discriminating these from each other is challenging at the molecular level
due to underlying stochastic behaviour. Analysis is traditionally performed
using mean square displacements of tracked particles, however, this generally
requires more data points than is typical for single FP tracks due to
photophysical instability. Presented here is a novel approach allowing robust
Bayesian ranking of diffusion processes (BARD) to discriminate multiple complex
modes probabilistically. It is a computational approach which biologists can
use to understand single molecule features in live cells.Comment: combined ms (1-37 pages, 8 figures) and SI (38-55, 3 figures
Myosin II filament dynamics in actin networks revealed with interferometric scattering microscopy
The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood due to difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering (iSCAT) microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect, and image individual membrane attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network
Motion Analysis of Live Objects by Super-Resolution Fluorescence Microscopy
Motion analysis plays an important role in studing activities or behaviors of live objects in medicine, biotechnology, chemistry, physics, spectroscopy, nanotechnology, enzymology, and biological engineering. This paper briefly reviews the developments in this area mostly in the recent three years, especially for cellular analysis in fluorescence microscopy. The topic has received much attention with the increasing demands in biomedical applications. The tasks of motion analysis include detection and tracking of objects, as well as analysis of motion behavior, living activity, events, motion statistics, and so forth. In the last decades, hundreds of papers have been published in this research topic. They cover a wide area, such as investigation of cell, cancer, virus, sperm, microbe, karyogram, and so forth. These contributions are summarized in this review. Developed methods and practical examples are also introduced. The review is useful to people in the related field for easy referral of the state of the art
Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM
Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research
Super-Resolution Microscopy: A Virus’ Eye View of the Cell
It is difficult to observe the molecular choreography between viruses and host cell components, as they exist on a spatial scale beyond the reach of conventional microscopy. However, novel super-resolution microscopy techniques have cast aside technical limitations to reveal a nanoscale view of virus replication and cell biology. This article provides an introduction to super-resolution imaging; in particular, localisation microscopy, and explores the application of such technologies to the study of viruses and tetraspanins, the topic of this special issue
Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus
Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.Nikon France (Research contract)France. Agence nationale de la recherche (PCV DYNAFT 08-PCVI-0013)France. Agence nationale de la recherche (DynamIC ANR-12-BSV5-0018)Fondation pour la recherche médicaleNetherlands Organization for Scientific Research (Rubicon fellowship
Transcription factor clusters regulate genes in eukaryotic cells
Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression
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