1,722,021 research outputs found
Spatiotemporal fluorescence correlation spectroscopy of inert tracers: a journey within cells, one molecule at a time
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In Vivo Imaging of Single-Molecule Translocation through Nuclear Pore Complexes by Pair Correlation Functions
Full text linkNuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. Although there are many studies that look at the traffic in the nucleus and through the nuclear envelope we propose a method to detect the nucleocytoplasmic transport kinetics in an unperturbed cell, with no requirement for specific labeling of isolated molecules and, most important, in the presence of the cell milieu.The pair correlation function method (pCF) measures the time a molecule takes to migrate from one location to another within the cell in the presence of many molecules of the same kind. The spatial and temporal correlation among two arbitrary points in the cell provides a local map of molecular transport, and also highlights the presence of barriers to diffusion with millisecond time resolution and spatial resolution limited by diffraction. We use the pair correlation method to monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which single particle tracking (SPT) has given results that cannot be confirmed by traditional single-point FCS measurements because of the lack of spatial resolution.We show that obstacles to molecular flow can be detected and that the pCF algorithm can recognize the heterogeneity of protein intra-compartment diffusion as well as the presence of barriers to transport across NE
Capturing directed molecular motion in the nuclear pore complex of live cells
Full text linkNuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleo-cytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center of mass of single NPCs. We find the dynamics of nucleoporin Nup153 to be regulated so as to produce rapid, discrete exchange between two separate positions within the NPC. A similar behavior is also observed for both karyopherinÎ21 transport-receptor and cargoes destined to nuclear import. Thus, we argue that directed Nup-mediated molecular motion may represent an intrinsic feature of the overall selective gating through intact NPCs
Super-Resolution by Feedback Imaging: Mechanisms of Translocation through the Nuclear Pore Complex
Full text linkExploring dynamics in living cells by tracking single particles
Full text linkIn the last years, significant advances in microscopy techniques and the introduction of a novel technology to label living cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biology. Our understanding on cell dynamics built from snapshots on fixed specimens has evolved thanks to our actual capability to monitor in real time the evolution of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components ranging from organelles to protein molecules by following their way through the cell. In this review, we introduce the single particle tracking technology to new users. We briefly describe the instrumentation and explain some of the algorithms commonly used to locate and track particles. Also, we present some common tools used to analyze trajectories and illustrate with some examples the applications of single particle tracking to study dynamics in living cells.Fil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Electrónica Cuántica; ArgentinaFil: Gratton, Enrico. University of California at Irvine; Estados Unido
Spatiotemporal Fluctuation Analysis: A Powerful Tool for the Future Nanoscopy of Molecular Processes
Full text linkAbstractThe enormous wealth of information available today from optical microscopy measurements on living samples is often underexploited. We argue that spatiotemporal analysis of fluorescence fluctuations using multiple detection channels can enhance the performance of current nanoscopy methods and provide further insight into dynamic molecular processes of high biological relevance
Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes.
No full textSpatial distribution and dynamics of plasma-membrane proteins are thought to be modulated by lipid composition and by the underlying cytoskeleton, which forms transient barriers to diffusion. So far this idea was probed by single-particle tracking of membrane components in which gold particles or antibodies were used to individually monitor the molecules of interest. Unfortunately, the relatively large particles needed for single-particle tracking can in principle alter the very dynamics under study. Here, we use a method that makes it possible to investigate plasma-membrane proteins by means of small molecular labels, specifically single GFP constructs. First, fast imaging of the region of interest on the membrane is performed. For each time delay in the resulting stack of images the average spatial correlation function is calculated. We show that by fitting the series of correlation functions, the actual protein "diffusion law" can be obtained directly from imaging, in the form of a mean-square displacement vs. time-delay plot, with no need for interpretative models. This approach is tested with several simulated 2D diffusion conditions and in live Chinese hamster ovary cells with a GFP-tagged transmembrane transferrin receptor, a well-known benchmark of membrane-skeleton-dependent transiently confined diffusion. This approach does not require extraction of the individual trajectories and can be used also with dim and dense molecules. We argue that it represents a powerful tool for the determination of kinetic and thermodynamic parameters over very wide spatial and temporal scales
Airyscan Cca Provides Structural and Dynamics Fingerprinting of Subcellular Compartments in Living Cells
No full textIn this work we will present our current advances in the development of the CCA (Comprehensive Correlation Analysis) technique using the Zeiss Airyscan detector. This detector consists of 32 GaAsP PMT arranged in a hexagonal pattern, featuring a fast temporal sampling (down to 1.28 μs per frame). It can also be used in a super-resolution configuration as per the ISM [1] principle, while the arrangement of the detectors allows for the implementation of advanced state-of-the-art spatiotemporal correlation techniques. We will present a novel application of the spot-variation FCS technique [2] in super-resolution, as well as the implementation of the 2D-pCF [3], iMSD [4] and number and brightness [5] analysis using this fast detector array. This simultaneous analysis of the same dataset provides biophysical information regarding the fluorescent probe and the surrounding environment, such as diffusion coefficient, concentration, diffusion modality, environment organization, direction and anisotropy of molecular flows, diffusion connectivity and oligomerization state in a single analysis in a few seconds
From fast fluorescence imaging to molecular diffusion law on live cell membranes in a commercial microscope
Full text linkIt has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn't need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on μm-sized membrane regions in the micro-to-milli-second time range
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