1,721,022 research outputs found
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
The Nucluear Pore Complex as Intrinsic Reporter for Isotropic Expansion Microscopy
No full textExpansion microscopy (ExM) is a super-resolution imaging method that does not require any special optical microscope(1). The key point resides on the possibility of uniformly chemically expanding a sample, thus increasing the relative distances among objects of interest as fluorescent molecules labeling specific components. ExM is highly invasive; it involves gelation and digestion steps that could introduce artifacts and heterogeneities in the relative spatial distribution of complex proteins in the cells. The possibility to combine STED(2) and ExM (ExSTED)(3) allows not only an unprecedented resolution but also a higher sensitivity to discover possible pitfalls. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters, i.e., scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC)(4). In particular, we show that Nup153, a filamentous subunit localized in the nuclear pore basket(5), is an excellent reporter to address the isotropy of the expansion process quantitatively. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale with consistent accuracy in the range of 20 nm. It is an excellent method for structural studies of macromolecular complexe
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
Fluorescence Correlation Spectroscopy of Intact Nuclear Pore Complexes
Full text linkAbstractNo methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-β1 (Kapβ1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kapβ1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions
Super-Resolution by Feedback Imaging: Mechanisms of Translocation through the Nuclear Pore Complex
Full text linkChromatin Nanoscale Organization Investigated by FLIM-FRET and STED Superresolution Microscopy
No full textInvestigation of Oncogene-Induced Alterations in Chromatin Organization In Vitro by Structured Illumination Microscopy
No full textOncogenes activation may lead to alterations in chromatin organization at the nanoscale by interfering with fundamental processes such as DNA transcription, DNA replication, and DNA Damage Response [1]. The in vitro observation of these molecular processes is crucial to understand how oncogenes can alter chromatin organization leading to tumor formation. Among all the available techniques to address this issue, optical super-resolution microscopy stands out for its potential in revealing the distribution of molecules within intact cell nuclei. Here we apply structured illumination microscopy (SIM)[2] to investigate a model of oncogene activation in an engineered cell line, U937-PR9, derived from Acute Promyelocytic Leukemia (APL) patients. In APL, 70% of the patients present a mutation that causes the formation of a fusion protein PML-RARalpha. This model allows us to selectively activate the PML-RAR ..
An Image-Based Approach to the Evaluation of Oncogene Activation Effects on Cell's Genomic Stability
No full textA Novel Viewpoint to Analyze Structured Illumination Microscopy (Sim) Data
No full textStructured Illumination Microscopy (SIM) is an important tool among the Super Resolution Microscopy techniques [1]. SIM stands out for its capability to perform optical sectioning, multicolor channel acquisition and live cell imaging, thanks to its low phototoxicity. In SIM, the specimen is illuminated with a high-frequency grating pattern, instead of a uniform field. The multiplication of sample features and this pattern gives rise to Moiré fringes.The obtained interference pattern allows encoding of sub-diffraction sample's features into lower frequencies collected by the objective lens. The reconstruction process then decodes otherwise unresolvable high-frequency information creating the super-resolved image.Here we aim to analyze SIM data from a rather different standpoint, based on the idea of Separation of Photons by LIfetime Tuning (SPLIT)[2]. In SPLIT the sub-diffraction spatial information is encoded into an additional channel of the microscope. The information encoded within this channel is visualized and analyzed in the phasor plot and allows separating a fractional image component of higher spatial resolution corresponding to the center of the PSF.In the context of Stimulated Emission Depletion (STED) microscopy, we recently demonstrated that SPLIT can be used to decode spatial information encoded in the nanosecond fluorescence lifetime [2] or variations of the depletion power [3]. We show here that in the case of SIM it is possible to use SPLIT to analyze the information encoded into the images acquired at different illumination patterns. In particular an important aspect of SPLIT is that the Phasor Plot provides a visual, intuitive and direct evaluation of the acquired data [3].We demonstrate that knowledge of the illumination pattern can be used to perform SPLIT analysis and to reconstruct a super-resolved image bypassing the traditional Fourier reconstruction.As an application, we use the SPLIT-SIM approach to perform super-resolution imaging of chromatin-related structures
Optimized Super-Resolution Imaging of Nuclear Sites in an Engineered Leukemia Cell Line
No full textStimulated emission depletion (STED) microscopy and Separation of Photons by Lifetime Tuning (SPLIT) provide optical super-resolution imaging, at the nanometer scale, on intact single cell nuclei. The resolution that can be achieved is limited by factors such as photobleaching and/or reduction of signal-to-noise, which depend on the conditions of image acquisition.
Here we use image correlation spectroscopy (ICS) to quantify, in an unbiased way, the resolution and signal-to-noise of STED and SPLIT images and determine how they are affected by multiple parameters such as photobleaching, STED power, and number of averages. Imaging conditions optimization is particularly relevant when it comes to analyzing genomic processes, such as DNA replication, occurring within the crowded nuclear environment. As an application, we optimize the imaging conditions on an engineered cell line, U937-PR9, derived ..
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